Methods and compositions for oral administration of insulin

ABSTRACT

The present invention provides a pharmaceutical composition formulated for oral delivery of insulin, comprising a particulate non-covalently associated mixture of pharmacologically inert silica nanoparticles having a hydrophobic surface, a polysaccharide, and insulin suspended in an oil. The present invention further provides methods of manufacturing same and therapeutic methods utilizing same for oral delivery of insulin.

FIELD OF INVENTION

The present invention relates to pharmaceutical compositions for oraldelivery of insulin, comprising an intimate mixture of solid dryparticulate ingredients within an oil. Specifically the pharmaceuticalcompositions comprise a particulate non-covalently associated intimatemixture of pharmacologically inert silica nanoparticles having ahydrophobic surface, a polysaccharide, and insulin suspended or embeddedin an oil or mixture of oils. The present invention further providesmethods of manufacturing same and therapeutic methods utilizing same fororal delivery of insulin.

BACKGROUND OF THE INVENTION

Oral delivery of active agents is a particularly desirable route ofadministration, because of safety and convenience considerations andbecause oral delivery replicates the physiologic mode of insulindelivery. In addition, oral delivery provides for more accurate dosingthan multidose vials and can minimize or eliminate the discomfort thatoften attends repeated hypodermic injections.

There are many obstacles to successful oral delivery of biologicalmacromolecules. For example, biological macromolecules are large and areamphipathic in nature. More importantly, the active conformation of manybiological macromolecules may be sensitive to a variety of environmentalfactors, such as temperature, oxidizing agents, pH, freezing, shakingand shear stress. In planning oral delivery systems comprisingbiological macromolecules as an active agent for drug development, thesecomplex structural and stability factors must be considered.

In addition, in general, for medical and therapeutic applications, wherea biological macromolecule is being administered to a patient and isexpected to perform its natural biological function, delivery vehiclesmust be able to release active molecules, at a rate that is consistentwith the needs of the particular patient or the disease process.

The hormone insulin, contributes to the normal regulation of bloodglucose levels through its release by the pancreas, more specifically bythe B-cells of a major type of pancreatic tissue (the islets ofLangerhans). Insulin secretion is a regulated process, which, in normalsubjects, provides stable concentrations of glucose in blood during bothfasting and feeding. Diabetes is a disease state in which the pancreasdoes not release insulin at levels capable of controlling glucoselevels. Diabetes is classified into two types. The first type isdiabetes that is insulin dependent and usually appears in young people.The islet cells of the pancreas stop producing insulin mainly due toautoimmune destruction and the patient must inject himself with themissing hormone. These Type 1 diabetic patients are the minority oftotal diabetic patients (up to 10% of the entire diabetic population).The second type of diabetes (type 2) is non-insulin dependent diabetes,which is caused by a combination of insulin resistance and insufficientinsulin secretion. This is the most common type of diabetes in theWestern world. Close to 8% of the adult population of various countriesaround the world, including the United States, have Type 2 diabetes, andabout 30% of these patients will need to use insulin at some pointduring their life span due to secondary pancreas exhaustion.

The problem of providing bioavailable unmodified human insulin, in auseful form, to the ever-increasing population of diabetics has occupiedphysicians and scientists for almost 100 years. Many attempts have beenmade to solve some of the problems of stability and biological deliveryof this small protein. Examples include: U.S. Pat. No. 7,455,830 whichdiscloses bioactive nanoparticles suitable for oral delivery of insulinwhich include a shell substrate of chitosan, and a core substrateselected from the group consisting of gamma-polyglutamic acid (PGA),alpha-PGA, water soluble salts of PGA and metal salts of PGA; U.S. Pat.No. 7,470,663 which discloses a liquid solution formulated for oraldelivery, comprising a substantially monodispersed mixture ofconjugates, wherein each conjugate comprises human insulin covalentlycoupled a carboxylic acid, which is covalently coupled at the end distalto the carboxylic acid moiety to a methyl terminated polyethylene glycolmoiety. U.S. Pat. No. 7,384,914 which discloses a method of treating amammal which has impaired glucose tolerance by administering atherapeutically effective dose of a pharmaceutical formulationcomprising insulin and the delivery agent4-[(2-hydroxy-4-chlorobenzoyl)amino]butanoate (4-CNAB) in an amountwhich facilitates absorption of the insulin from the gastrointestinaltract of the treated mammal, and U.S. Pat. No. 6,656,922 which disclosesa method for enhancing oral administration of insulin by conjugatinginsulin to an hydrophobic agent selected from the group consisting ofbile acids, sterols, alkanoic acids, and mixtures thereof to result in ahydrophobized macromolecular agent.

As of today, most diabetic patients self-administer insulin by dailysubcutaneous injections. However, the limitations of multiple dailyinjections, such as inconvenience, poor patient acceptability,compliance and the difficulty of matching postprandial insulinavailability to postprandial requirements are some of the better knownshortcomings of insulin therapy.

A method of providing insulin without the need for injections has been agoal in drug delivery. Insulin absorption in the gastrointestinal tractis prevented by its large size and enzymatic degradation. It would bedesirable to create an oral pharmaceutical formulation of a drug such asinsulin (which is not normally orally administrable due to, e.g.,insufficient absorption from the gastrointestinal tract), whichformulation would provide sufficient absorption andpharmacokinetic/pharmacodynamic properties to provide the desiredtherapeutic effect.

Accordingly, there is a need for a method of administering insulin topatients in need of insulin wherein those patients are not subject tosystemic hyperinsulinemia, which by itself can increase the risk ofvascular disease (that is normally associated with such chronic insulintreatments, as discussed above). In other words, it is desirable toprovide compositions and methods for treating diabetes without thedrawbacks of systemic hyperglycemia to decrease the incidence ofvascular complications and other detrimental effects.

Biopolymers and their Use in Delivering Active Proteins Such as Insulin:

Biopolymers such as polysaccharides have been known for many years.Polysaccharides are widely used as excipients in oral dosage forms, asdisclosed for example in U.S. Pat. No. 7,351,741 to Weidner, U.S. Pat.No. 6,667,060 to Vandecruys and US patent application 2004/0115264 toBlouquin. These references neither disclose nor suggest use ofbiopolymers in combination with nanoparticles and/or oil.

Nanoparticles and their Use in Delivering Active Proteins Such asInsulin:

Silica nanoparticles are well known in the art as pharmaceuticalexcipients and are their use is disclosed for example in U.S. Pat. Nos.6,322,765 to Muhlhofer and 6,698,247 to Tennent, among many others.Coating of a nanoparticle-biopolymer complex with oil, or utility ofsame in oral administration of insulin are neither disclosed norsuggested.

Methods for imparting a hydrophobic surface to nanoparticles are wellknown in the art and are described, for example, in Chung et al.(Hydrophobic modification of silica nanoparticle by using aerosol sprayreactor. Colloids and Surfaces A: Physicochem. Eng. Aspects 236 (2004)73-79). Additional methods include the reverse micelles method (Fu X,Qutubuddin S, Colloids Surf. A: Physicochem. Eng. Aspects 179: 65,2001), liquid precipitation method (Krysztafkiewicz A, Jesionowski T,Binkowski S, Colloids Surf. A: Physicochem. Eng. Aspects 173:73, 2000)and sol-gel method (Jean J, Yang S, J. Am. Ceram. Soc. 83 (8):1928,2000; Zhang J, Gao L, Ceram. Int. 27: 143, 2001). Use of nanoparticlesin combination with a biopolymer and insulin, coating ananoparticle-containing complex with oil, or utility of same in oraladministration of insulin are neither disclosed nor suggested.

U.S. Pat. Nos. 7,105,229, 6,989,195, 6,482,517, 6,638,621, 6,458,387,7,045,146, and 5,462,866 among many others disclose use of nanoparticlesor microparticles as excipients for proteins. These references neitherdisclose nor suggest intimate non-covalent association of nanoparticleswith a biopolymer and insulin, or embedding of ananoparticle-biopolymer-insulin in an oil coating.

US 2007/0154559 to Pai discloses an orally administrable compositioncontaining nanoparticles comprising a charged water-soluble drug incomplex with a counter-ion substance, a lipid, a polymer, and anemulsifier. The compositions are formed by (a) ionically bonding thedrug with the counter-ion; (b) adding a lipid, a polymer, and asolubilizing agent; dissolving the whole mixture; and introducing thesolution into an aqueous solution containing an emulsifier; and (c)removing the solubilizing agent. US 2006/0177495 and 2003/0235619 toAllen disclose delivery vehicles for delivering an active agent,comprising nanoparticles composed of a biodegradable hydrophobic polymerforming a core and an outer amphiphilic layer surrounding the polymercore and containing a stabilizing lipid.

US 2006/0083781 to Shastri discloses nanoparticles comprising a lipidand a polymer comprising an ionic or ionizable moiety. Thesecompositions as well differ significantly from those of the presentinvention, inter alia in that (a) the polymer is not outside thenanoparticles but rather forms a part of them; and (b) the oil forms apart of the nanoparticles instead of coating the nanoparticle-polymermixture. In addition, the unique structures of the matrix carriercompositions of the present invention is neither disclosed norsuggested.

WO 96/37232 to Alonso Fernandez discloses methods for preparation ofcolloidal systems through the formation of ionic lipid-polysaccharidecomplexes. The colloidal systems are stabilized through the formation ofan ionic complex, at the interface, comprised of a positively chargedaminopolysaccharide and a negatively charged phospholipid. Thesecompositions as well differ significantly from those of the presentinvention, inter alia in that (a) the polymer is not outside thenanoparticles but rather forms a part of them; and (b) the oil forms apart of the nanoparticles instead of coating them. In addition, use ofinsulin as an active agent is neither disclosed or suggested.

U.S. Pat. No. 6,548,264 to Tan et al. discloses silica-coatednanoparticles and a process for producing silica-coated nanoparticles.Silica-coated nanoparticles are prepared by precipitating nano-sizedcores from reagents dissolved in the aqueous compartment of awater-in-oil microemulsion. A reactive silicate is added to coat thecores with silica. The silicate coating may further be derivatized witha protein. US 2007/0275969 to Gurny discloses pharmaceuticalcompositions for the oral administration of pharmaceutical agents havinglow water solubility. The pharmaceutical agents are solubilized with apolymer, from which nanoparticles are formed.

In cosmetics formulations, it is common to use compositions comprisingwater-in-oil emulsions containing an aqueous phase dispersed in an oilyphase. There are numerous examples in which silica nanoparticles as wellas polysaccharides are included in the liquid fatty phase. U.S. Pat. No.6,228,377 for example, discloses water-in-oil emulsions containing aliquid fatty phase which contains hydrophobic or hydrophilic fumedsilica, a branched polysaccharide alkyl ether, an emulsifying surfactantand oil. These compositions differ significantly from those of thepresent invention in that they include a water phase and surfactantsthat serve as the most important structure forming factor of thecomposition.

Additional Strategies

Methods for oral administration of insulin are the object of extensiveresearch efforts but have been proven generally inefficient to date. Anumber of strategies for preventing degradation of orally administeredproteins have been suggested, including use of core-shell particles(U.S. Pat. No. 7,090,868 to Gower) and nano-tubes (U.S. Pat. No.7,195,780 to Dennis). Liposomes have been used as a carrier for orallyadministered proteins, as well as aqueous emulsions and suspensions(U.S. Pat. No. 7,316,818; WO 06/062544; U.S. Pat. No. 6,071,535; andU.S. Pat. No. 5,874,105 to Watkins) and gas-filled liposomes (U.S. Pat.No. 6,551,576; U.S. Pat. No. 6,808,720; and U.S. Pat. No. 7,083,572 toUnger et al.). Another composition comprises nanodroplets dispersed inan aqueous medium (US 2007/0184076). Additional strategies are found inWO 06/097793, WO 05/094785, and WO 03/066859 to Ben-Sason, whichdescribe matrix-carriers containing peptide-effectors that providepenetration across biological barriers for administration of hydrophobicproteins; and EP 0491114B1 to Guerrero Gomez-Pamo, which describespreparation of non-covalent protein-polysaccharide complexes for oraladministration of biologically active substances, stabilized byprecipitates of organic salts. None of these references discloses orsuggests intimate non-covalent association of nanoparticles with abiopolymer or a nanoparticles-polymer matrix embedded in an oil coating.

In addition to the differences outlined above, none of the abovereferences discloses or suggests the enhanced bioavailability ofcompositions of the present invention.

SUMMARY OF THE INVENTION

The present invention relates to pharmaceutical compositions for oraldelivery of insulin, comprising an intimate mixture of solid dryparticulate ingredients within an oil. Preferably the compositions areanhydrous. Specifically the pharmaceutical compositions comprise aparticulate non-covalently associated mixture of pharmacologically inertsilica nanoparticles having a hydrophobic surface, a polysaccharide, andinsulin suspended in, embedded in or dispersed in an oil or mixture ofoils. The present invention further provides methods of manufacturingsame and therapeutic methods utilizing same for oral delivery ofinsulin.

According to the present invention it is now disclosed for the firsttime that the compositions of the invention surprisingly enable oralbioavailability of insulin. The present invention is based in part onthe surprising discovery that experimental diabetic mice treated orallywith a composition of the present invention, maintained normal bloodglucose levels (˜100 mg/dL) for up to 12 hours after administration ofthe drug whereas diabetic mice given the same amount of insulin byintravenous injection could not maintain a normal blood glucose levelfor over 6 hours.

In one aspect, the present invention provides a pharmaceuticalcomposition for oral delivery of insulin comprising an oil havingparticulate matter suspended therein, wherein the particulate matterincludes: (a) pharmacologically inert silica nanoparticles having ahydrophobic surface, wherein the size of the nanoparticles is between1-100 nanometers, in intimate non-covalent association with apolysaccharide; and (b) an insulin protein attached to the silicananoparticles and the polysaccharide via non-covalent forces. In anotherembodiment, the insulin protein is attached to the hydrophobic surfacesof the silica nanoparticles and the polysaccharide via non-covalentforces (FIG. 1). In another embodiment, the hydrophobic portion of theinsulin protein is attached to the hydrophobic surfaces of the silicananoparticles and the polysaccharide via non-covalent forces. In anotherembodiment, the hydrophilic portion of the insulin protein is alsonon-covalently attached to hydrophilic portion of the polysaccharide. Inanother embodiment, the non-covalent forces cause the nanoparticles,polysaccharide, and insulin to form an intimate mixture. Eachpossibility represents a separate embodiment of the present invention.

In one preferred embodiment of the present invention, the polysaccharidecomprises a branched polysaccharide. In another embodiment, the branchedpolysaccharide is selected from the group consisting of amylopectin,starch and glycogen. In another embodiment, the branched polysaccharideis starch.

In another embodiment, the particulate matter including the hydrophobicsilica nanoparticles, the polysaccharide and the insulin is dispersedin, embedded in or suspended within the oil phase of the matrixcomposition. In another embodiment, the oil phase is impregnated withthe particulate matter. As provided herein, the present inventionprovides compositions wherein the particulate matter form a matrix thatis impregnated and completely surrounded by the oil phase. Preferablythe weight of polysaccharides will be greater than the weight of thesilica. In some embodiments the weight of the polysaccharides will be atleast twice that of the silica, in other embodiments the weight of thepolysaccharides will be 5 fold that of the silica in yet otherembodiments the polysaccharides will be at least 10 times greater thanthe weight of silica nanoparticles. Each possibility represents aseparate embodiment of the present invention.

Reference to silica nanoparticles of the present invention as having a“hydrophobic” surface encompasses silica nanoparticles having a surfacemodified to be hydrophobic. In another embodiment, the silicananoparticles are modified by chemically coating the surface with ahydrocarbon. In another embodiment, the coating causes the silicananoparticles to display hydrocarbon moieties on their surface. Methodsfor imparting a hydrophobic surface to silica nanoparticles are wellknown in the art, and are described inter alia herein. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, a pharmaceutical composition of the presentinvention comprises a mixture of oils selected from natural vegetableoils and synthetic analogues thereof.

In another embodiment, a pharmaceutical composition of the presentinvention further comprises an additional oil component. The term“additional oil component” encompasses an additional oil or mixture ofoils, as described elsewhere herein. In another embodiment, theadditional oil component comprises an antioxidant. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, a pharmaceutical composition of the presentinvention further comprises a third oil or mixture of oils in additionto the above-described additional oil or mixture of oils. In anotherembodiment, the third oil component comprises an antioxidant. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, a matrix composition of the present inventionfurther comprises a wax.

In another embodiment, a matrix composition formulated for oraladministration of the present invention is in the form of a tablet,capsule, or suspension.

In another embodiment, a pharmaceutical composition of the presentinvention further comprises an additional biopolymer that is a linearbiopolymer. In another embodiment, the additional biopolymer is apolysaccharide. In another embodiment, the additional biopolymer is alinear polysaccharide. In another embodiment, the additional biopolymeris a linear high molecular weight structural protein. In anotherembodiment, the additional biopolymer is selected from the groupconsisting of chitin, cellulose, a linear alpha glucan, and a linearbeta glucan. In another embodiment, the additional biopolymer isselected from the group consisting of chitin, amylose, cellulose, andbeta glucan. In another embodiment, a pharmaceutical composition of thepresent invention comprises a branched polysaccharide and a linearpolysaccharide. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the additional biopolymer of methods andcompositions of the present invention is a fiber. In another embodiment,the fiber is a dietary fiber. In another embodiment, the dietary fiberis an insoluble fiber. In another embodiment, the dietary fiber is alinear insoluble fiber. In another embodiment, the dietary fiber is asoluble fiber. In another embodiment, the dietary fiber is a linearsoluble fiber. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, a pharmaceutical composition of the presentinvention comprises a branched biopolymer, a linear polysaccharide, andan insoluble fiber. In another embodiment, a composition of the presentinvention comprises a branched polysaccharide, a linear polysaccharide,and an insoluble fiber. An example of such is a composition comprisingamylopectin, a branched polysaccharide; chitin, a linear polysaccharide;and cellulose, an insoluble fiber. Other branched and linearpolysaccharides and insoluble fibers disclosed herein are suitable aswell. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the present invention provides a method ofadministering an insulin protein to a subject in need thereof,comprising orally administering to the subject a pharmaceuticalcomposition of the present invention, thereby administering an insulinprotein to a subject.

In another embodiment, the present invention provides a method oftreating diabetes in a subject in need thereof, comprising orallyadministering to the subject a pharmaceutical composition of the presentinvention, thereby treating diabetes in a subject. In anotherembodiment, the diabetes is an insulin-dependent diabetes. In anotherembodiment, the diabetes is a non-insulin-dependent diabetes. In anotherembodiment, the diabetes is Type I diabetes. In another embodiment, thediabetes is Type II diabetes. In another embodiment, the diabetes isjuvenile diabetes. In another embodiment, the diabetes is adolescentdiabetes. In another embodiment, the diabetes is adult diabetes. Inanother embodiment, the diabetes is any other type of diabetes known inthe art. In another embodiment, a method of the present invention isused to treat a complication of diabetes. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the subject of a method of the present inventionis a human. In another embodiment, the subject is a non-human mammal.Each possibility represents a separate embodiment of the presentinvention.

As provided herein, oral administration of compositions of the presentinvention lowers blood glucose levels for several hours in animal(Example 6) and human (Example 7) subjects, without causing glycemicinstability or troublesome hypoglycemia symptoms. Further, thecompositions exhibit no detectable toxicity (Example 8).

In another embodiment, the present invention provides use of apharmaceutical composition of the present invention in the preparationof a medicament for administering insulin to a subject.

In another embodiment, the present invention provides use of apharmaceutical composition of the present invention in the preparationof a medicament for treating diabetes in a subject.

In another embodiment, the present invention provides a pharmaceuticalcomposition of the present invention for administering insulin to asubject.

In another embodiment, the present invention provides a pharmaceuticalcomposition of the present invention for treating diabetes in a subject.

In certain embodiments, the insulin in a pharmaceutical composition ofthe present invention is capable of reaching the bloodstream of asubject, following oral administration, with over 20% of the biologicalactivity intact, preferably over 30% of the biological activity remainsintact, more preferably at least 40% of the biological activity remainsintact, most preferably at least 50% of the biological activity remainsintact. In another embodiment, over 60% of the biological activityremains intact. In another embodiment, over 70% of the biologicalactivity remains intact. In another embodiment, over 80% of thebiological activity remains intact. In another embodiment, over 90% ofthe biological activity remains intact. Without wishing to be bound byany theory or mechanism of action, these properties are believed to bedue to protection of the active agent from digestive enzymes andmechanical forces in the intestines by the excipients of pharmaceuticalcompositions of the present invention.

In another embodiment, a pharmaceutical composition of the presentinvention is designed to provide short-term release. “Short-termrelease”, as used herein, refers to release over 8-12 hours, withmaximal activity 4 hours after administration. In another embodiment, apharmaceutical composition of the present invention is designed toprovide medium-term release. “Medium-term release”, as used herein,refers to release over 12-18 hours, with maximal activity 4-6 hoursafter administration. In another embodiment, a pharmaceuticalcomposition of the present invention is designed to provide long-termrelease. “Long-term release”, as used herein, refers to release over18-48 hours, with maximal activity 4-8 hours after administration. Inanother embodiment, a pharmaceutical composition of the presentinvention is designed to provide very long-term release. “Very long-termrelease”, as used herein, refers to release over 18-72 hours, withmaximal activity 6-8 hours after administration. In another embodiment,the longer term-release compositions of the present invention exhibit alower peak with a longer tail following the peak activity. Eachpossibility represents a separate embodiment of the present invention.

In another aspect, the present invention provides a method ofmanufacturing a pharmaceutical composition for oral delivery of insulin,the method comprising the steps of: (a) blending pharmacologically inertsilica nanoparticles having a hydrophobic surface, wherein the size ofthe nanoparticles is between 1-100 nanometers, with a polysaccharide,whereby the silica nanoparticles form an intimate non-covalentassociation with the polysaccharide; (b) mixing an insulin protein withan oil; and (c) mixing the nanoparticles and polysaccharide into theoil. In another embodiment, the silica nanoparticles, polysaccharide,and insulin thereby form a matrix that becomes dispersed, embedded orsuspended in the oil. Preferably, the silica nanoparticles,polysaccharide, and insulin form a complex. In another embodiment, thecomplex is dispersed, embedded or suspended in the oil. In anotherembodiment, the insulin protein is non-covalently attached to thehydrophobic surfaces of the silica nanoparticles and to the hydrophilicand hydrophobic portions, regions or patches of the surface of thepolysaccharide. In another embodiment, the particle size of thepharmaceutical composition is between 100-500,000 nanometers (nm). Insome preferred embodiments, the particle size is between 100-50,000 nm.Each possibility represents a separate embodiment of the presentinvention.

In yet another aspect, the present invention provides a method ofmanufacturing a pharmaceutical composition for oral delivery of insulin,the method comprising the steps of: (a) blending pharmacologically inertsilica nanoparticles having a hydrophobic surface, wherein the size ofthe nanoparticles is between 1-100 nanometers, with (i) apolysaccharide, and (ii) an insulin protein whereby the silicananoparticles form an intimate non-covalent association with thepolysaccharide; and (b) mixing the particulate matter (silicananoparticles, polysaccharide, and insulin protein) into an oil. Inanother embodiment, the silica nanoparticles, polysaccharide, andinsulin form a matrix that becomes dispersed, embedded or suspended inthe oil. Preferably, the silica nanoparticles, polysaccharide, andinsulin form a complex. In another embodiment, the complex is dispersed,embedded or suspended in the oil. In another embodiment, the insulinprotein is non-covalently attached to the hydrophobic surfaces of thesilica nanoparticles and to the hydrophilic and hydrophobic portions,regions or patches of the surface of the polysaccharide. In anotherembodiment, the particle size of the pharmaceutical composition isbetween 100-500,000 nanometers. In some preferred embodiments, theparticle size is between 100-50,000 nanometers. Each possibilityrepresents a separate embodiment of the present invention. As providedherein, methods have been developed to formulate insulin in orallyadministrable form. In certain preferred embodiments, the components aremixed in a particular order in order to produce oil-coated matrixcarrier compositions that protect the insulin from digestive processesin the stomach and small intestine. Further, without wishing to be boundby any theory or mechanism of action, the polysaccharide, particularlywhen branched, absorbs hydraulic and mechanical stresses experiencedduring digestion. The oil coating constitutes a physical barrier thatprovides additional protection against digestive enzymes. Thepharmaceutical compositions of the present invention are converted inthe digestive system to particles smaller in size but similar instructure to the original composition, which are absorbed similarly tochylomicrons and reach the bloodstream without undergoing first-passmetabolism in the liver. In another embodiment, the particles are brokendown in gastro-intestinal tract to particles having a characteristicsize between 30-1000 nanometers. In certain preferred embodiments, thesize of the particles after digestion is between 30-700 nm. While theprimary particles are in the nanometer to sub-micrometer range, thesemay form conglomerates or agglomerates of larger dimensions within thecompositions of the present invention. The size of these conglomeratesor agglomerates ranges between 100-500,000 nanometers. In some preferredembodiments, the conglomerate or agglomerate size is between 100-50,000nanometers. In another embodiment, the conglomerate or agglomerate sizeis between 100-5,000 nanometers. Each possibility represents a separateembodiment of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic view of a representative matrix-carrier structurecontaining insulin, silica nanoparticles and a polysaccharide. Top:Macrostructure containing branched fiber structure of the polysaccharideimpregnated with hydrophobic silica nanoparticles. Bottom:microstructure depiction.

FIG. 2: Schematic view of the structure formed in the small intestinedue to joint action of hydrodynamic and enzymatic processes.

FIG. 3: Light microscopy picture of insulin matrix carrier FormulationIV (Example 5).

FIG. 4: A. Effect of oral administration of NovoRapid™ insulin oralcomposition of the present invention (Formulation VI, Example 4) onblood glucose levels (BGL) in diabetic (STZ-treated) mice. Differentsymbols represent individual mice. The break indicates the time of theinsulin composition administration. B. Effect of oral administration ofActrapid™ insulin compositions (Formulation V, Example 4) on BGL indiabetic (STZ-treated) mice. Different symbols represent individualmice.

FIG. 5: BGL levels in STZ-treated mice orally receiving 25 IU Insulin(by BIOCON) in PBS (gavage). Different symbols represent individualmice.

FIG. 6: A. Dose response curve towards the insulin matrix carriercompositions (Formulation IV, Example 5) of the present invention on STZmice (mean blood glucose concentrations are based on the BGL of at least5 mice). B-D. Data from individual STZ mice administered (FormulationIV): 2 (B), 5 (C), and 10 (D) IU of insulin. E. Effect of SC-injectedinsulin on STZ mice. F. Effect of 12 IU of insulin composition on normalmice. The breaks in figures B-I indicate the time of administration.Different symbols represent individual mice. G, H, I: Comparison of theeffect of 10 (G), 5 (H), and 2 (I) IU of insulin on the BGL uponadministration of insulin by SC injection and orally using the matrixcarrier composition of the present invention. J-K. Comparison of thepharmacodynamics of two different matrix carrier compositions for oraldelivery of insulin (formulation A versus formulation IV) —2 IU ofinsulin (J) and 7.5 IU of insulin (K).

FIG. 7: Efficacy of oral insulin compositions of the present inventionon healthy (A) and diabetic (B) human subjects. A. 30 IU of theActrapid™ relatively short-term release insulin matrix carriercomposition (Formulation II) was administered at time 12:00, asindicated by the stripe in the graph. B. Daily average blood glucoselevels. Gluco-Rite™ was administered on days 2-12. Insulin matrixcarrier composition (formulation V, example 4) was first administered onthe 13^(th) day and continued for 14 days.

FIG. 8: Toxicity study of insulin oral compositions (Formulation IV) ofthe present invention. Microscopic analysis of the liver (A); kidney(B); and duodenum (C). In each case, left panels are control samples andright panels are treated samples.

FIG. 9: A-D Cryo SEM freeze fracture pictures of mice serum taken 4hours after oral administration (gavage) of 10 IU oral insulincomposition (Formulation IV).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides matrix carrier compositions for oraldelivery of insulin, comprising an intimate mixture of solid dryparticulate ingredients within an oil. Specifically the pharmaceuticalcompositions comprise a particulate non-covalently associated mixture ofpharmacologically inert silica nanoparticles having a hydrophobicsurface, a branched polysaccharide and insulin suspended, embedded ordispersed in an oil or mixture of oils. The present invention furtherprovides methods of manufacturing same and therapeutic methods utilizingsame for oral delivery of insulin.

The oral insulin compositions of the present invention provide anadvantageous result over the subcutaneously administered insulin, whichis currently the state of the art, beyond the benefit of ease ofadministration, pain-free administration, and the potential for improvedpatient compliance. By administration of the oral insulin compositionsof the present invention, the blood levels of insulin which occur uponthe first (initial) phase of insulin secretion by the pancreas can besimulated. The first phase of insulin secretion, while of shortduration, has an important role in priming the liver to the metabolicevents ahead (meal). Because subcutaneously administered insulin doesnot undergo portal circulation, this result is not possible withsubcutaneously administered insulin.

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising: (a) pharmacologically inert silica nanoparticleshaving a hydrophobic surface, wherein the diameter of the nanoparticlesis between 1-100 nanometers, in intimate mixture with a polysaccharide;and (b) an insulin protein non-covalently attached to the silicananoparticles and the polysaccharide; wherein the matrix formed by thesilica nanoparticles, polysaccharide, and insulin is suspended, embeddedor dispersed in oil. In another embodiment, the insulin isnon-covalently attached to the hydrophobic surfaces of the silicananoparticles and to the hydrophilic and hydrophobic portions, regionsor patches of the surface of the polysaccharide. In another embodiment,the hydrophobic portion of the insulin protein is attached to thehydrophobic surfaces of the silica nanoparticles and the polysaccharidevia non-covalent forces. In another embodiment, the hydrophilic portionof the insulin protein is also non-covalently attached to hydrophilicportion of the polysaccharide. In another embodiment, the particlediameter of the pharmaceutical composition following its formation, butprior to ingestion is between 100-500,000 nm. In certain preferredembodiments, the particle diameter is between 100-50,000 nanometers. Inanother embodiment, the particle diameter is between 100-5000 nm. Eachpossibility represents a separate embodiment of the present invention.

Various components of pharmaceutical compositions of the presentinvention, namely insulin, silica nanoparticles, polysaccharides, highmolecular weight structural proteins, and oils, are described herein.Each embodiment thereof can be utilized in methods of the presentinvention, and each such use represents a separate embodiment of thepresent invention.

In another embodiment, the oil phase of the matrix carrier compositioncomprises a plurality of oils.

In another embodiment, a pharmaceutical composition of the presentinvention is held together by non-covalent forces (FIG. 1). In anotherembodiment, without wishing to be bound by any theory or mechanism ofaction, the non-covalent forces between the components of the matrixcomposition enable the matrix composition to self-assemble when thecomponents are mixed together, as described herein. In anotherembodiment, the non-covalent forces cause the silica nanoparticles,polysaccharide and insulin to form an intimate mixture. In anotherembodiment, the matrix composition exhibits an ordered structure. Inanother embodiment, without wishing to be bound by any theory ormechanism of action, the matrix composition includes a solid phasecontaining at least two solid pharmacologically inert materials (silicananoparticles and polysaccharides) with different properties. In anotherembodiment, the silica nanoparticle/polysaccharide/insulin complex isdispersed, embedded or suspended within the oil phase of the matrixcomposition. In another embodiment, the oil phase is impregnated withthe silica nanoparticle/polysaccharide/insulin complex of the matrixcomposition. As provided herein, the present invention providescompositions wherein the silica nanoparticles, polysaccharide, andinsulin form a matrix that is impregnated and completely surrounded bythe oil phase. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, a pharmaceutical composition of the presentinvention further comprises an additional biopolymer that is a linearbiopolymer. In another embodiment, the additional biopolymer is a linearpolysaccharide. In another embodiment, the additional biopolymer is alinear high molecular weight structural protein. In another embodiment,the additional biopolymer is selected from the group consisting ofchitin, cellulose, a linear alpha glucan, and a linear beta glucan. Inanother embodiment, the additional biopolymer is selected from the groupconsisting of chitin, amylose, cellulose, and beta glucan. Insulincompositions are provided herein that comprise amylopectin, a branchedbiopolymer, and chitin, a linear biopolymer (Example 5). Other branchedand linear biopolymers disclosed herein are suitable as well. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the additional biopolymer of methods andcompositions of the present invention is a dietary fiber. In anotherembodiment, the dietary fiber is an insoluble fiber. In anotherembodiment, the dietary fiber is a linear insoluble fiber. In anotherembodiment, the dietary fiber is a soluble fiber. In another embodiment,the dietary fiber is a linear soluble fiber.

In another embodiment, a composition of the present invention comprisesa branched biopolymer, a linear polysaccharide, and an insoluble fiber.In another embodiment, a composition of the present invention comprisesa branched biopolymer, a polypeptide, and an insoluble fiber. An exampleof such is a composition comprising amylopectin, a branchedpolysaccharide; keratin, a polypeptide; and cellulose, an insolublefiber. Other branched polysaccharides, polypeptides, and insolublefibers disclosed herein are suitable as well. In another embodiment, acomposition of the present invention comprises a branchedpolysaccharide, a linear polysaccharide, and an insoluble fiber. Anexample of such is a composition comprising amylopectin, a branchedpolysaccharide; chitin, a linear polysaccharide; and cellulose, aninsoluble fiber. Other branched and linear polysaccharides and insolublefibers disclosed herein are suitable as well. Each possibilityrepresents a separate embodiment of the present invention.

Oil having particulate matter suspended therein, as used herein, refersto particulate matter that is in contact with oil. The composition as awhole need not be homogeneous with regard to the distribution of theparticulate matter. Rather, the particulate matter is capable of beingdispersed or suspended in the oil when agitated. The particulate matterneed not be completely homogeneous, but rather is characterized by itscontaining the ingredients specified herein and its intimate contactwith the oil of the present invention. Compositions wherein theparticulate matter is agglomerated fall within the scope of the presentinvention.

Nanoparticles

The silica nanoparticles of methods and compositions of the presentinvention are preferably pharmacologically inert. In another embodiment,the silica nanoparticles are composed of materials that are generallyrecognized as safe (GRAS). In another embodiment, the silicananoparticles are non-toxic. In another embodiment, the silicananoparticles are non-teratogenic. In another embodiment, the silicananoparticles are biologically inert. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the nanoparticles are silica-containingnanoparticles. “Silica-containing nanoparticles” refers preferably tonanoparticles comprising silica, a silicate, or a combination thereof.“Silica” refers to silicon dioxide. Silica-containing nanoparticles areavailable commercially, e.g. as 99.99% pure finely ground silica. Itwill be understood by those skilled in the art that lower grades ofpurity of silica are also compatible with the present invention.“Silicate” refers to a compound containing silicon and oxygen, e.g. intetrahedral units of SiO₄. In another embodiment, the term refers to acompound containing an anion in which one or more central silicon atomsare surrounded by electronegative ligands. Non-limiting examples ofsilicates are hexafluorosilicate, sodium silicate (Na₂SiO₃), aluminumsilicates, magnesium silicates, etc. It is to be understood that thenanoparticles in structures of the present invention can be either of asingle type or of multiple types, provided that, if multiple types arepresent, at least one type is a silica-containing nanoparticles. Inanother embodiment, essentially all the nanoparticles aresilica-containing nanoparticles. Silica is widely recognized as a safefood additive (Thirteenth report of the Joint FAO/WHO Expert Committeeon Food Additives, FAO Nutrition Meetings Report Series; from the JointFAO/WHO Expert Committee on Food Additives meeting in Rome, May 27-Jun.4, 1969). Each possibility represents a separate embodiment of thepresent invention.

Reference to silica nanoparticles of the present invention as having a“hydrophobic” surface indicates, in one embodiment, that at least 40% ofthe silica nanoparticle surface is hydrophobic. In another embodiment,at least 50% of the surface is hydrophobic. In another embodiment, atleast 60% of the surface is hydrophobic. In another embodiment, at least70% of the surface is hydrophobic. In another embodiment, at least 80%of the surface is hydrophobic. In another embodiment, at least 90% ofthe surface is hydrophobic. In another embodiment, at least 95% of thesurface is hydrophobic. In another embodiment, 40-100% of the surface ishydrophobic. In another embodiment, 50-100% of the surface ishydrophobic. In another embodiment, 60-100% of the surface ishydrophobic. In another embodiment, 70-100% of the surface ishydrophobic. In another embodiment, 80-100% of the surface ishydrophobic. In another embodiment, 90-100% of the surface ishydrophobic. In another embodiment, 95-100% of the surface ishydrophobic. In another embodiment, 40-60% of the surface ishydrophobic. In another embodiment, 40-50% of the surface ishydrophobic. In another embodiment, 40-70% of the surface ishydrophobic. In another embodiment, 40-80% of the surface ishydrophobic. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, reference to silica nanoparticles as having a“hydrophobic” surface encompasses silica nanoparticles having a surfacechemically modified to be hydrophobic. In another embodiment, thenanoparticles are chemically modified by coating the surface with ahydrocarbon. In another embodiment, the coating causes the nanoparticlesto display hydrocarbon moieties on their surface. In another embodiment,the hydrocarbon moieties are selected from the group consisting ofmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, T-butyl, pentyl, andiso-pentyl. In another embodiment, the coating causes the nanoparticlesto display methyl moieties on their surface. Methods for imparting ahydrophobic surface to nanoparticles are well known in the art, and aredescribed inter alia herein. As is known in the art it is possible tochemically modify the surface of the fumed silica by chemical reaction,generating a decrease in the number of silanol groups. In particular,silanol groups can be substituted with hydrophobic groups to obtain ahydrophobic silica. The hydrophobic groups can be: trimethylsiloxygroups, which are obtained in particular by treatment of fumed silica inthe presence of hexamethyldisilazane. Silicas thus treated are known as“silica silylate” according to the CTFA (6th edition, 1995). They aresold, for example, under the references “Aerosil R812®” by the companyDegussa and “CAB-OSIL TS-530®” by the company Cabot; dimethylsilyloxy orpolydimethylsiloxane groups, which are obtained in particular bytreatment of fumed silica in the presence of polydimethylsiloxane ordimethyldichlorosilane. Silicas thus treated are known as “silicadimethyl silylate” according to the CTFA (6th edition, 1995). They aresold, for example, under the references “Aerosil R972®.”, “AerosilR974®” by the company Degussa, “CAB-O-SIL TS-610®.” and “CAB-O-SILTS-720®.” by the company Cabot. Each possibility represents a separateembodiment of the present invention.

In another embodiment, nanoparticles of compositions of the presentinvention are practically insoluble in water. “Practically insoluble”refers, in another embodiment, to a substance having a solubility ofless than 100 parts per million weight/weight (ppm). In anotherembodiment, the term refers to a solubility of less than 200 ppm. Inanother embodiment, the term refers to a solubility of less than 80 ppm.In another embodiment, the term refers to a solubility of less than 60ppm. In another embodiment, the term refers to a solubility of less than50 ppm. In another embodiment, the term refers to a solubility of lessthan 40 ppm. In another embodiment, the term refers to a solubility ofless than 30 ppm. In another embodiment, the term refers to a solubilityof less than 20 ppm. In another embodiment, the term refers to asolubility of less than 15 ppm. In another embodiment, the term refersto a solubility of less than 10 ppm. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the diameter of the silica nanoparticles ofmethods and compositions of the present invention is between 5-30nanometers inclusive. In another embodiment, the diameter is between2-400 nanometers (nm) inclusive. In another embodiment, the diameter isbetween 2-300 nm inclusive. In another embodiment, the diameter isbetween 3-200 nm inclusive. In another embodiment, the diameter isbetween 4-150 nm inclusive. In another embodiment, the diameter isbetween 4-100 nm inclusive. In another embodiment, the diameter isbetween 5-50 nm inclusive. In another embodiment, the diameter isbetween 5-40 nm inclusive. In another embodiment, the diameter isbetween 6-25 nm inclusive. In another embodiment, the mean diameter ofsilica nanoparticles is 10-11 nm.

In another embodiment, the average diameter is about 5 nm. In anotherembodiment, the average diameter is about 6 nm. In another embodiment,the average diameter is about 7 nm. In another embodiment, the averagediameter is about 8 nm. In another embodiment, the average diameter isabout 9 nm. In another embodiment, the average diameter is about 10 nm.In another embodiment, the average diameter is about 12 nm. In anotherembodiment, the average diameter is about 14 nm. In another embodiment,the average diameter is about 16 nm. In another embodiment, the averagediameter is about 18 nm. In another embodiment, the average diameter isabout 20 nm. In another embodiment, the average diameter is anotherdiameter falling within a range disclosed herein. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, silica nanoparticles of the present inventionfall within a range of melting temperatures particularly suitable forcompositions of the present invention. In specific embodiments, thesilica nanoparticles have a melting temperature (T_(m)) of over 600° C.In another embodiment, the T_(m) is between 600-4500° C. In anotherembodiment, the T_(m) is another T_(m) falling within a range disclosedherein. Each possibility represents a separate embodiment of the presentinvention.

Imparting a Hydrophobic Surface to a Nanoparticle

Methods for imparting a hydrophobic surface to nanoparticles are wellknown in the art and are described, inter alia, in Chung et al(Hydrophobic modification of silica nanoparticle by using aerosol sprayreactor. Colloids and Surfaces A: Physicochem. Eng. Aspects 236 (2004)73-79). Additional methods include the reverse micelles method (Fu X,Qutubuddin S, Colloids Surf. A: Physicochem. Eng. Aspects 179: 65,2001), liquid precipitation method (Krysztafkiewicz A, Jesionowski T,Binkowski S, Colloids Surf. A: Physicochem. Eng. Aspects 173:73, 2000)and sol-gel method (Jean J, Yang S, J. Am. Ceram. Soc. 83 (8):1928,2000; Zhang J, Gao L, Ceram. Int. 27: 143, 2001). US 2007/0172426provides additional methods of imparting a hydrophobic surface to ananoparticle, by combining them with a material having a first end thatadsorbs to the surface of the nanoparticle and a second end that extendsaway from the nanoparticle and imparts hydrophobicity to the particles.The material may be a generally aliphatic compound having a polarend-group. The first end of each molecule of the compound may include acarboxyl group, an amine group, a silane, etc., that adsorbs to thesurface of the particle. The second end of each molecule of the compoundmay include alkane group that extends away from the particle. Materialsused to provide the hydrophobic surface layer include saturated fattyacids such as lauric acid, myristic acid, palmitic acid, and stearicacid, and unsaturated variants thereof, such as palmitoleic acid, oleicacid, linoleic acid, and linolenic acid. Silanes such as octadecyltrichlorosilane can also be widely used to functionalize oxide surfaces.The hydrophobic surface layer is provided by mixing the nanoparticlesinto a volume of hydrophobic coating material suitable for coating theparticles. An excess of hydrophobic coating material is generally usedso that the nanoparticles form a suspension in the hydrophobic coatingmaterial. Each nanoparticle then exhibits a hydrophobic layer on itssurface. Additional methods for utilizing a hydrocarbon surfactant tocoat nanoparticles are described in US 2006/0053971. Additional methodsare described in US 2007/0098990. The disclosed methods utilize multipleorganic acids in which the first acid is a low molecular weight organiccarboxylic acid and the second acid is a high molecular weight organiccarboxylic acid. The contents of each of the above patent applicationsare hereby incorporated by reference.

Biopolymers

Methods and compositions of the present invention is preferably comprisea branched biopolymer. “Branched” as used herein encompasses bothpolymers that are naturally branched and those engineered to be branchedby physical treatment such as thermal and/or ultrasound treatment. Ingeneral, branched polymers are defined as polymers wherein a monomersubunit is covalently bound to more than two monomer subunits. Such amonomer is the site of a branch point, wherein multiple polymer chainsconverge. In another embodiment, the branched biopolymer is acrosslinked polymer. In another embodiment, the branched biopolymer isnot crosslinked. Non-limiting examples of branched polymers are glycogenand amylopectin, forms of starch found in animals and plants,respectively. Structures of amylopectin (CAS#9037-22-3) and glycogen(CAS#9005-79-2) are depicted below:

In another embodiment, the biopolymer is a fibrous biopolymer. “Fibrouspolymer” refers to a polymer in the form of a network of discretethread-shaped pieces. Non-limiting examples of fibrous polymers are guargum (found for example in Benefiber™), collagen, keratin, fibrin, andelastin. Biopolymers can be either naturally fibrous or made fibrous byphysical and chemical treatment.

In another embodiment, the biopolymer is a fiber. “Fiber” refers, inanother embodiment, to an indigestible component that acts as a bulkingagent for feces. In another embodiment, the fiber is an insoluble fiber.In another embodiment, the fiber is a soluble fiber. Each possibilityrepresents a separate embodiment of the present invention. Each type offiber and type of branched and fibrous biopolymer represents a separateembodiment of the present invention.

In another embodiment, the biopolymer is pharmacologically inert. Inanother embodiment, the biopolymer is non-toxic. In another embodiment,the biopolymer is non-teratogenic. In another embodiment, the biopolymeris biologically inert. Each possibility represents a separate embodimentof the present invention.

In another embodiment, the melting temperature of the biopolymer fallswithin a range particularly suitable for compositions of the presentinvention. In another embodiment, the biopolymer has a meltingtemperature under 400° C. In another embodiment, the T_(m) is below 350°C. In another embodiment, the T_(m) is below 300° C. In anotherembodiment, the T_(m) is below 250° C. In another embodiment, the T_(m)is below 200° C. In another embodiment, the T_(m) is below 150° C. Inanother embodiment, the T_(m) is between 100-400° C. In anotherembodiment, the T_(m) is any T_(m) falling within a range disclosedherein. Each possibility represents a separate embodiment of the presentinvention.

Preferably, the biopolymer of methods and compositions of the presentinvention is selected from the group consisting of a polysaccharide anda structural protein.

Polysaccharides

“Saccharide” refers to any simple carbohydrate includingmonosaccharides, monosaccharide derivatives, monosaccharide analogs,sugars, including those, which form the individual units in apolysaccharide. “Monosaccharide” refers to polyhydroxyaldehyde (aldose)or polyhdroxyketone (ketose) and derivatives and analogs thereof.

“Polysaccharide” refers to polymers formed from about 500 saccharideunits linked to each other by hemiacetal or glycosidic bonds. Typically,polysaccharides can contain as many as 100,000 saccharide units, and insome cases even more. The polysaccharide may be either a straight chain,singly branched, or multiply branched wherein each branch may haveadditional secondary branches, and the monosaccharides may be standardD- or L-cyclic sugars in the pyranose (6-membered ring) or furanose(5-membered ring) forms such as D-fructose and D-galactose,respectively, or they may be cyclic sugar derivatives, for example aminosugars such as D-glucosamine, deoxy sugars such as D-fucose orL-rhamnose, sugar phosphates such as D-ribose-5-phosphate, sugar acidssuch as D-galacturonic acid, or multi-derivatized sugars such asN-acetyl-D-glucosamine, N-acetylneuraminic acid (sialic acid), orN-sulfato-D-glucosamine. When isolated from nature, polysaccharidepreparations comprise molecules that are heterogeneous in molecularweight. Polysaccharides include, among other compounds, galactomanansand galactomannan derivatives; galacto-rhamnogalacturons andgalacto-rhamnogalacturon derivatives, and galacto-arabinogalacturon andgalacto-arabinogalacturon derivatives.

In another embodiment, the polysaccharide of methods and compositions ofthe present invention is a naturally-occurring polysaccharide. Inanother embodiment, the polysaccharide is a synthetic polysaccharide.Non limiting examples of synthetic polysaccharides can be found in U.S.Pat. No. 6,528,497 and in Okada M. et al. Polymer journal, 15 (11);821-26 (1983). In another embodiment, the polysaccharide is anaturally-occurring branched polysaccharide. In another embodiment, thepolysaccharide is a synthetic branched polysaccharide. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the polysaccharide is a branched polysaccharide.This term is well understood to those skilled in the art and can referto any number and structure of branches in the links betweenmonosaccharide monomers. In another embodiment, the polysaccharide is anaturally-occurring branched polysaccharide. In another embodiment, thebranched polysaccharide is a starch. In another embodiment, the branchedpolysaccharide is selected from the group consisting of amylopectin,glycogen, and a branched alpha glucan. In another embodiment, thepolysaccharide is a synthetic branched polysaccharide. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the polysaccharide is an amphipathicpolysaccharide. This term is well understood to those skilled in the artand refers to the existence of both hydrophobic and hydrophilic regionson the polysaccharide. In another embodiment, the polysaccharide is anaturally-occurring amphipathic polysaccharide. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the average MW of the polysaccharide is at least100 kilodalton (kDa). In another embodiment, the average MW is at least150 kDa. In another embodiment, the average MW is at least 200 kDa. Inanother embodiment, the average MW is at least 300 kDa. In anotherembodiment, the average MW is at least 400 kDa. In another embodiment,the average MW is at least 500 kDa. In another embodiment, the averageMW is at least 600 kDa. In another embodiment, the average MW is atleast 800 kDa. In another embodiment, the average MW is at least 1000kDa. In another embodiment, the average MW is between 100-1000 kDa. Inanother embodiment, the average MW is between 150-1000 kDa. In anotherembodiment, the average MW is between 200-1000 kDa. In anotherembodiment, the average MW is between 100-800 kDa. In anotherembodiment, the average MW is between 100-600 kDa. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the polysaccharide is selected from the groupconsisting of starch, dextrin, cellulose, chitin, a branched alphaglucan, a branched beta glucan and derivatives thereof. Cellulose,dextrin, starch and glycogen are all polymers of glucose and thus havethe formula (C₆H₁₀O₅)_(n).

In another embodiment, the polysaccharide is a starch, which has thestructure below. Non-limiting examples of starch are corn starch, potatostarch, rice starch, wheat starch, purum starch, and starch from algae.In another embodiment, the starch is any other starch known in the art.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the polysaccharide is a dextrin. “Dextrin” inanother embodiment refers to a low-molecular-weight carbohydrateproduced by the hydrolysis of starch. In another embodiment, the termrefers to a linear α-(1,4)-linked D-glucose polymer starting with anα-(1,6) bond or a mixture of same. Dextrins are widely commerciallyavailable and can be produced inter alia by digestion of branchedamylopectin or glycogen with α-amylase. A non-limiting example of adextrin is a maltodextrin having the structure below. In anotherembodiment, the dextrin is any other dextrin known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the polysaccharide is cellulose. A non-limitingexample of a cellulose is α-cellulose, which has the structure below.

In another embodiment, the polysaccharide is β-cellulose, a linearpolymer of D-glucose linked by β(1→4) glycosidic bonds. In anotherembodiment, the β-cellulose has the structure below.

In another embodiment, the cellulose is any other cellulose known in theart. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the polysaccharide is chitin, a long-chainpolymer of N-acetylglucosamine, a derivative of glucose. Typically,chitin has the molecular formula (C₈H₁₃NO₅)_(n) and the structure below.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the polysaccharide is an alpha-glucan.Alpha-glucans of the present invention may be linear or branchedpolymers of glucose with alpha 1-2, alpha 1-3, alpha 1-4, and/or alpha1-6 glycosidic linkages. In another embodiment, the alpha-glucan hasunbranched linear glucose polymers with 1-4 glycosidic linkages, anexample of which is alpha-amylose. In another embodiment, thealpha-glucan has branched glucose polymers with alpha 1-4 glycosidiclinkages in the backbone and alpha 1-6 linkages at branch points, anexample of which is amylopectin. In another embodiment, the alpha-glucanis any other type of alpha-glucan known in the art. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the polysaccharide is a beta-glucan.“Beta-glucan” refers to polysaccharides containing D-glucopyranosylunits linked together by (1→3) or (1→4) beta-linkages. Beta-Glucansoccur naturally in many cereal grains such as oats and barley. Themolecular weight of beta-glucan molecules occurring in cereals istypically 200 to 2000 kDa; other types contain up to about 250,000glucose units. In another embodiment, the beta-glucan is any otherbeta-glucan known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, a pharmaceutical composition of the presentinvention comprises a branched polysaccharide and a linearpolysaccharide. In another embodiment, the linear polysaccharide isselected from the group consisting of chitin, cellulose, amylose, andbeta glucan. In some preferred embodiments, the branched and linearpolysaccharides both have a melting temperature under 400° C. Insulincompositions are provided herein that comprise amylopectin, a branchedpolysaccharide, and chitin, a linear polysaccharide (Example 5,Formulation IV). Other branched polysaccharides and linearpolysaccharides disclosed herein are suitable as well.

In another embodiment, the additional biopolymer of methods andcompositions of the present invention is a fiber, preferentially adietary fiber. The definition of the term “fiber” and “dietary fiber” asused herein includes unavailable carbohydrates, indigestible residue,and plant cell polysaccharides and lignin, all of which are resistant tohydrolysis by human digestive enzymes. Preferred fibers are membersselected from the group consisting of guar gum, pectin,fructo-oligosaccharides and derivatives thereof. Small amounts of otherindigestible compounds, such as phytates, tannins, saponins and cutin,may be included in dietary fiber since these compounds are indigestibleand associated with dietary fiber polysaccharides. In anotherembodiment, the dietary fiber is an insoluble fiber. In anotherembodiment, the dietary fiber is a linear insoluble fiber. In anotherembodiment, the dietary fiber is a soluble fiber. In another embodiment,the dietary fiber is a linear soluble fiber. Each possibility representsa separate embodiment of the present invention.

In another embodiment, the T_(m) of a polysaccharide of a composition ofthe present invention falls within a range of melting temperaturesparticularly suitable for compositions of the present invention. Inanother embodiment, the polysaccharide has a T_(m) under 400° C. Inanother embodiment, the T_(m) is another T_(m) or range of T_(m) definedherein. Each possibility represents a separate embodiment of the presentinvention.

Structural Proteins

According to certain embodiments the dry solid particulate ingredientsof compositions may further comprise a structural protein. thestructural protein of methods and compositions of the present inventionis a high molecular weight (MW) structural protein. In some embodiments,the structural protein comprises both hydrophilic and hydrophobicresidues that interact with the hydrophobic and hydrophilic regions,respectively, of the biologically active protein or peptide. In anotherembodiment, the average MW of the structural protein is at least 100kilodalton (kDa). In another embodiment, the average MW is at least 150kDa. In another embodiment, the average MW is at least 200 kDa. Inanother embodiment, the average MW is at least 300 kDa. In anotherembodiment, the average MW is at least 400 kDa. In another embodiment,the average MW is at least 500 kDa. In another embodiment, the averageMW is at least 600 kDa. In another embodiment, the average MW is atleast 800 kDa. In another embodiment, the average MW is at least 1000kDa. In another embodiment, the average MW is between 100-1000 kDa. Inanother embodiment, the average MW is between 150-1000 kDa. In anotherembodiment, the average MW is between 200-1000 kDa. In anotherembodiment, the average MW is between 100-800 kDa. In anotherembodiment, the average MW is between 100-600 kDa. Each possibilityrepresents a separate embodiment of the present invention.

“Structural protein”, in one embodiment, refers to a protein includedfor the structure it confers to the matrix carrier composition. Inanother embodiment, a structural protein of the present invention lackstherapeutic activity. In another embodiment, the term refers to aprotein that confers structure to a cell, cellular membrane, orextracellular membrane in vivo. In another embodiment, the structuralprotein is a fibrous protein. In another embodiment, the structuralprotein is a scleroprotein. In another embodiment, the structuralprotein is selected from the group consisting of elastin, collagen,keratin, and fibrinogen. In another embodiment, the structural proteinis any other fibrous protein or scleroprotein known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the structural protein is elastin. Non-limitingexamples of elastin proteins are described, inter alia, in GenBankAccession numbers NP_(—)031951, NP_(—)786966, and AAC98394. In anotherembodiment, the elastin is any other elastin known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the structural protein is collagen. Non-limitingexamples of collagen proteins include those encoded by gene symbolsCOL3A1, COL14A1, COL11A2, COL5A2, COL11A1, COL5A1, COL4A6, COL4A5,COL4A4, COL4A3, COL4A2, COL1A2, COL5A3, COL18A1, COL12A1, COL19A1,COL24A1, COL4A1, and COL2A1. In another embodiment, the collagen is anyother collagen known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the structural protein is keratin. Non-limitingexamples of keratin proteins include keratin 18, keratin 14, keratin 3,and keratin 86 (GenBank Accession numbers P05783, P02533, P12035,O43790, respectively. In another embodiment, the keratin is any otherkeratin known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the structural protein is fibrinogen. Fibrinogenis a glycoprotein composed of three pairs of polypeptides: two alpha,two beta, and two gamma chains. Non-limiting examples of the fibrinogenalpha, beta, and gamma chains are described, inter alia, in GenBankAccession numbers P02671, P02675, and P02679. In another embodiment, thefibrinogen is any other fibrinogen known in the art. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the T_(m) of a structural protein of acomposition of the present invention falls within a range of meltingtemperatures particularly suitable for compositions of the presentinvention. In another embodiment, the structural protein has a T_(m)under 400° C. Each possibility represents a separate embodiment of thepresent invention.

Oils and Oil Coatings

The particulate matter of matrix compositions of the present inventionis surrounded by, suspended in, immersed in, embedded in or dispersed inoil carrier. Typically, the oil phase, in addition to coating theparticulate matter, impregnates the particulate matter, which iscomposed of the silica nanoparticles, branched polysaccharide andinsulin. Reference to an “oil,” “oil layer,” “oil phase,” or “oilcoating” does not preclude the presence of an additional component orcomponents useful in methods of the present invention (e.g. afat-soluble co-factor or anti-oxidant). Rather, the term indicates thatthe oil, oil layer, oil phase, or coating is composed primarily of apharmaceutically acceptable oil carrier, in which the other componentsare mixed and/or dissolved. The oil carrier can be composed of eitherone or a plurality of types of oils, as described further herein. Inanother embodiment, the coating consists essentially of lipids and/oroils. In another embodiment, the coating of the composition comprises apharmaceutically acceptable oil carrier. In another embodiment, the oilcarrier is a naturally occurring oil. In another embodiment, the oil isa mixture of natural vegetable oils. In another embodiment, the oilcarrier is sesame oil. In another embodiment, the oil carrier is oliveoil. In another embodiment, the oil carrier is linseed oil. In anotherembodiment, the oil carrier is evening primrose oil. In anotherembodiment, the oil carrier is silicone oil. In another embodiment, theoil carrier is sea buckthorn oil. In another embodiment, the oil carrieris selected from the group consisting of sesame oil, olive oil, linseedoil, evening primrose oil, silicone oil, and sea buckthorn oil. Inanother embodiment, the oil carrier includes, but is not limited to, anoil selected from the group consisting of sunflower oil, corn oil,soybean oil, jojoba oil, marrow oil, grapeseed oil, hazelnut oil,apricot oil, macadamia oil and castor oil. In another embodiment, theoil carrier is another suitable oil known in the art. In anotherembodiment, the oil carrier is of animal origin, such as lanolin. Inanother embodiment, the oil carrier is a synthetic oil. In anotherembodiment, the oil carrier is a fatty alcohol. In certain preferredembodiments, the oil carrier is 2-octyldodecanol. In certain otherpreferred embodiments, the oil carrier is selected from the groupconsisting of a fatty acid ester and a phenylsilicone. In certain morepreferred embodiments, the oil carrier is selected from the groupconsisting of a phenyltrimethicone, a diphynyldimethicone, and apoly-methylphenylsiloxane. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the oil consists essentially ofnaturally-occurring lipids and/or oils. Each possibility represents aseparate embodiment of the present invention.

“Plurality of oils” refers, in another embodiment, to two or more oils.In another embodiment, a composition of the present invention comprisesthree or more oils. In another embodiment, a composition of the presentinvention comprises four or more oils. In another embodiment, acomposition of the present invention comprises more than four oils. Inanother embodiment, the oil phase comprises a mixture of oils selectedfrom natural vegetable oils. Each possibility represents a separateembodiment of the present invention.

In another embodiment, an oil component of the present inventioncomprises a component capable of stimulating secretion of bile salts orbile acids when ingested by a subject. In another embodiment, thebile-stimulating component is an oil. In another embodiment, thecomponent is olive oil or an extract thereof. In another embodiment, thecomponent is any other bile salt/acid stimulating lipid-solublesubstance known in the art. In another embodiment, the carrier is thebile salt/acid stimulating substance. In another embodiment, the bilesalt/acid stimulating substance is a substance separate from thecarrier. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, an oil component of the present inventioncontains a significant quantity of one or more anti-oxidants. Forexample, sea buckthorn (oblepicha) oil contains a significant quantityof beta-carotene. In another embodiment, any other oil enriched in oneor more anti-oxidants may be used. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, an oil component of the present inventioncomprises a component that has a melting temperature (T_(m)) of at least10° C. In another embodiment, the high T_(m) component is an oil. Inanother embodiment, the carrier is the high T_(m) component. In anotherembodiment, the high-T_(m) component is included in addition to thecarrier. A non-limiting example of a high-T_(m) oil is jojoba oil. Inanother embodiment, the high T_(m) oil is any other high meltingtemperature oil known in the art. In another embodiment, the high T_(m)oil is used as the oil carrier in the first oil component of a matrixcarrier of the present invention. Each possibility represents a separateembodiment of the present invention.

In another embodiment, a matrix composition of the present inventionfurther comprises a third oil or mixture of oils. In another embodiment,the third oil component comprises an antioxidant. In another embodiment,the third oil component is sesame oil. In another embodiment, the thirdoil component is another suitable oil known in the art. In anotherembodiment, the third oil, oil or mixture of oils has a higher viscositythan the additional oil or mixture of oils. Each possibility representsa separate embodiment of the present invention.

In another embodiment, a matrix composition of the present inventionfurther comprises an additional oil component. As provided herein,mixing of multiple oil components of compositions of the presentinvention in the correct order provides self-ordering orself-organization of matrix structure, due to competitive adsorption andminimization of the free energy. The term “additional oil component”encompasses an oil or mixture of oils, as described elsewhere herein. Inanother embodiment, the oil carrier of the additional oil component isolive oil. In another embodiment, the oil carrier is another suitableoil known in the art. In another embodiment, the additional oilcomponent comprises an antioxidant. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the insulin protein is included in the additionaloil or mixture of oils, instead of in the first-added oil or mixture ofoils. In another embodiment, the insulin protein is combined with anantioxidant and oil (the first-added or additional oil or mixture ofoils) prior to adding to the solid phase. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the additional oil, oil or mixture of oils has ahigher viscosity than the first-added oil or mixture of oils. In anotherembodiment, without wishing to be bound by any theory or mechanism ofaction, the use of a higher viscosity oil or oil mixture at this stageenables self-ordering or self-organization of structure due tocompetitive adsorption and minimization of the free energy. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, a matrix composition of the present inventionfurther comprises a third oil or mixture of oils. In another embodiment,the third oil component comprises an antioxidant. In another embodiment,the oil carrier of the third oil component is sesame oil. In anotherembodiment, the oil carrier is another suitable oil known in the art. Inanother embodiment, the third oil, oil or mixture of oils has a higherviscosity than the additional oil or mixture of oils. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, a highly penetrative oil carrier is included inthe outer oil or mixture of oils. Non-limiting examples of highlypenetrative oils are sesame oil, tea tree (Melaleuca) oil, lavender oil,almond oil, and grape seed oil. In another embodiment, the highlypenetrative oil carrier promotes efficient transport of the substancesinto the blood. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, a matrix composition or pharmaceuticalcomposition of the present invention further comprises apharmaceutically acceptable wax. The term “wax” means a lipophiliccompound, which is solid at room temperature (25° C.), with a reversiblesolid/liquid change of state, having a melting point of greater than orequal to 30° C., which may be up to 120° C. By bringing the wax to theliquid state (melting), it is possible to render it miscible with anyoils present and to form a microscopically homogeneous mixture, but onreturning the temperature of the mixture to room temperature,recrystallization of the wax in the oils of the mixture is obtained. Thewax may be a natural wax, for example bees wax, a wax derived from plantmaterial, or a synthetic wax prepared by esterification of a fatty acidand a long chain alcohol. Other suitable waxes include petroleum waxessuch as a paraffin wax. In another embodiment, the wax stabilizes thematrix carrier composition. In another embodiment, the inclusion of waxfacilitates formation of a tablet containing the matrix carriercomposition. Each possibility represents a separate embodiment of thepresent invention.

Insulin Proteins

“Insulin protein” as used herein includes rapid-acting insulin, veryrapid-acting insulin, intermediate-acting insulin, and long-actinginsulin. Non-limiting examples of rapid-acting insulin are lysproinsulin (Lysine-Proline insulin, sold by Eli Lilly as Humalog™),glu-lysine insulin (sold by Sanofi-Aventis as Apidra™), Actrapid™ andNovoRapid™ (both available from Novo Nordisk), aspart insulin (sold byNovo Nordisk as Novolog™). A non-limiting example of very rapid-actinginsulin is Viaject™, marketed by Biodel. Non-limiting examples ofintermediate-acting insulin are NPH (Neutral Protamine Hagedorn) andLente insulin. A non-limiting example of long-acting insulin is Lantus™(insulin glargine). In some preferred embodiments, the insulin isInsugen™ from Biocon™. In another embodiment, the insulin is a mixtureof different types of insulin. Some non-limiting examples of a such amixture are Mixtard® 30, Mixtard® 40, and Mixtard® 50, which aremixtures of different proportions of short-acting insulin and NPH(intermediate duration) insulin. In another embodiment, the insulin isany other type of insulin known in the art. In another embodiment, theinsulin is naturally occurring insulin. In another embodiment, theinsulin is a modified form of insulin. It will be clear from the presentdisclosure that methods and compositions of the present invention aresuitable for every type of natural and modified insulin known in theart. Each possibility represents a separate embodiment of the presentinvention.

Additional Components

In another embodiment, the composition further comprises an antioxidant.In another embodiment, the antioxidant is a pharmaceutically acceptableantioxidant. In another embodiment, the antioxidant is selected from thegroup consisting of vitamin E, superoxide dismutase (SOD), omega-3, andbeta-carotene. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the composition further comprises an enhancer ofthe insulin protein. Non limiting examples of insulin enhancers include:dodecylmaltoside, octylglucoside, and dioctyl sodium sulphosuccinate. Inanother embodiment, the composition further comprises a cofactor of theinsulin protein. Non limiting example of an insulin cofactor ischromium. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, a composition of the present invention furthercomprises a glucagon-like peptide or glucagon-like peptide analogue.Glucagon-like peptides and their analogues are well known in the art,and are described, inter alia, in Eleftheriadou I. et al. (The effectsof medications used for the management of diabetes and obesity onpostprandial lipid metabolism. Curr Diabetes Rev 4 (4):340-56, 2008 andVaidya H B et al., Glucagon like peptides-1 modulators as newer targetfor diabetes. Curr. Drug Targets 9 (10):911-20, 2008). Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, a composition of the present invention furthercomprises a bioflavonoid. Bioflavonoids are well known in the art, andare described, inter alia, in Ververidis F. et al. (Biotechnology offlavonoids and other phenylpropanoid-derived natural products. Part I:Chemical diversity, impacts on plant biology and human health and PartII: Reconstruction of multienzyme pathways in plants and microbes, 2(10):1214-49, 2007). Each possibility represents a separate embodimentof the present invention.

In another embodiment, a composition of the present invention furthercomprises a pharmaceutical-grade surfactant. Surfactants are well knownin the art, and are described, inter alia, in the Handbook ofPharmaceutical Excipients (eds. Raymond C. Rowe, Paul J. Sheskey, andSian C. Owen, copyright Pharmaceutical Press, 2005). In anotherembodiment, the surfactant is any other surfactant known in the art.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, a composition of the present invention furthercomprises pharmaceutical-grade emulsifier or emulgator (emollient).Emulsifiers and emulgators are well known in the art, and are described,inter alia, in the Handbook of Pharmaceutical Excipients (ibid).Non-limiting examples of emulsifiers and emulgators are eumulgin,Eumulgin B1 PH, Eumulgin B2 PH, hydrogenated castor oil cetostearylalcohol, and cetyl alcohol. In another embodiment, the emulsifier oremulgator is any other emulsifier or emulgator known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, a composition of the present invention furthercomprises pharmaceutical-grade stabilizer. Stabilizers are well known inthe art, and are described, inter alia, in the Handbook ofPharmaceutical Excipients (ibid). In another embodiment, the stabilizeris any other stabilizer known in the art. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the weight of the particulate matter of acomposition of the present invention is not more than 33% of the weightof the oil phase. The particulate matter is composed of the silicananoparticles, the branched polysaccharide, the insulin, and any othersolid components that may be incorporated into the matrix. In anotherembodiment, the particulate matter is composed of the silicananoparticles, the branched polysaccharide, and the insulin. The weightof the particulate matter is the weight of the oil carrier plusadditional oils mixed therewith and substances dissolved therein, ifany, for all the oil components combined. In another embodiment, theweight of the particulate matter is not more than 30% of the weight ofthe oil phase. In another embodiment, the weight of the particulatematter is not more than 25% of the weight of the oil phase. In anotherembodiment, the weight of the particulate matter is not more than 20% ofthe weight of the oil phase. In another embodiment, the weight of theparticulate matter is not more than 15% of the weight of the oil phase.In another embodiment, the weight of the particulate matter is not morethan 10% of the weight of the oil phase. In another embodiment, theweight of the particulate matter is not more than 8% of the weight ofthe oil phase. In another embodiment, the weight of the particulatematter is not more than 5% of the weight of the oil phase. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the weight of the particulate matter is not morethan 75% of the total weight of the composition. In another embodiment,the weight of the particulate matter is not more than 50% of the totalweight of the composition. In another embodiment, the weight of theparticulate matter is not more than 25% of the total weight of thecomposition. In another embodiment, the weight of the particulate matteris not more than 30% of the total weight of the composition. In anotherembodiment, the weight of the particulate matter is not more than 20% ofthe total weight of the composition. In another embodiment, the weightof the particulate matter is not more than 15% of the total weight ofthe composition. In another embodiment, the weight of the particulatematter is not more than 10% of the total weight of the composition. Inanother embodiment, the weight of the particulate matter is not morethan 8% of the total weight of the composition. In another embodiment,the weight of the particulate matter is not more than 6% of the totalweight of the composition. In another embodiment, the weight of theparticulate matter is not more than 5% of the total weight of thecomposition. Each possibility represents a separate embodiment of thepresent invention.

Methods of Administration

In another embodiment, the present invention provides a method ofadministering an insulin protein to a subject in need thereof,comprising orally administering to the subject a pharmaceuticalcomposition of the present invention, thereby administering an insulinprotein to a subject.

Formulation Methods

In another embodiment, the present invention provides a method ofmanufacturing a pharmaceutical composition for oral delivery of insulin,the method comprising the steps of: (a) dry blending pharmacologicallyinert silica nanoparticles having a hydrophobic surface, wherein thesize of the silica nanoparticles is between 1-100 nanometers, with atleast one branched polysaccharide, whereby the silica nanoparticles forman intimate non-covalent association with the at least one branchedpolysaccharide; (b) mixing or dissolving an insulin protein into an oil;and (c) mixing the silica nanoparticles and at least one branchedpolysaccharide into the oil, wherein the silica nanoparticles, at leastone branched polysaccharide, and insulin are suspended in, embedded inor dispersed in the oil. Preferably, the silica nanoparticles and atleast one branched polysaccharide form a complex. In another embodiment,the complex is suspended in, embedded in or dispersed in the oil. Inanother embodiment, the insulin protein is attached to the hydrophobicsurfaces of the silica nanoparticles and the at least one branchedpolysaccharide via non-covalent forces. In another embodiment, theparticle size of the matrix carrier composition is between 100-500,000nanometers. In some preferred embodiments, the particle size is between100-50,000 nanometers. In another embodiment, the particle size isbetween 100-5,000 nm. Each possibility represents a separate embodimentof the present invention.

Formulation methods of the present invention encompass embodimentswherein additional components are present in step (a). In anotherembodiment, more than one type of biopolymer is present together withthe silica nanoparticles. In another embodiment, a branchedpolysaccharide and a dietary fiber are present together with the silicananoparticles. In another embodiment, a branched polysaccharide and alinear polysaccharide are present together with the silicananoparticles. In another embodiment, a branched biopolymer, a linearpolysaccharide, and an insoluble fiber are present together with thesilica nanoparticles.

In another embodiment, the present invention provides a method ofmanufacturing a pharmaceutical composition for oral delivery of insulin,the method comprising the steps of: (a) blending pharmacologically inertsilica nanoparticles having a hydrophobic surface, wherein the size ofthe silica nanoparticles is between 1-100 nanometers, with (i) at leastone branched polysaccharide and (ii) an insulin protein whereby thesilica nanoparticles form an intimate non-covalent association with theat least one branched polysaccharide; and (b) mixing the silicananoparticles, at least one branched polysaccharide, and insulin proteininto an oil. In certain embodiments, the insulin protein in the form ofa dry lyophilized powder is directly dissolved into the oil of step (b).Preferably, the silica nanoparticles, at least one branchedpolysaccharide, and insulin form a complex. In another embodiment, thesilica nanoparticles, branched polysaccharide, and insulin form a matrixthat becomes suspended in, embedded in or dispersed in the oil. Inanother embodiment, the insulin protein is non-covalently attached tothe hydrophobic surfaces of the silica nanoparticles and the at leastone branched polysaccharide. In another embodiment, the particle size ofthe pharmaceutical composition is between 100-500,000 nanometers. Insome preferred embodiments, the particle size is between 100-50,000nanometers. In other embodiments, the particle size is between 100-5,000nanometers. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the insulin is extracted from an aqueoussolution. In another embodiment, an aqueous insulin solution is mixedwith oil, resulting in extraction or dispersion of the insulin directlyinto the oil phase of the resulting emulsion. Methods for extractingactive enzymes such as insulin from an aqueous solution are well knownin the art. In another embodiment, a gel-forming water phase stabilizeris used to extract the insulin. A non-limiting example of a gel-formingwater phase stabilizer is Silica 380, which is available as pharma-gradehydrophilic nanoparticles. Each possibility represents a separateembodiment of the present invention.

In another embodiment, step (b) of the above method comprises the stepof directly dissolving or dispersing a lyophilized protein into the oilor oil mixture. In another embodiment, a solution of the insulin proteinis mixed with the oil or oil mixture and the aqueous phase is thenremoved. In another embodiment, a solution of the insulin protein ismixed with the oil or oil mixture forming water-in-oil emulsion. Eachpossibility represents a separate embodiment of the present invention.

The properties and classification of the silica nanoparticles, branchedpolysaccharide, and insulin protein of the above methods may be any ofthose described herein. Each possibility represents a separateembodiment of the present invention. “Oil” as referred to in methods ofthe present invention can refer to either a single oil, a mixture ofoils, or an oil phase. As described herein, a mixture of oils or oilphase will typically comprise an oil carrier. In another embodiment, themixture of oils or oil phase further comprises an additional oil or oilsor an additional component or components. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, step (a) of a method of manufacturing apharmaceutical composition for oral delivery of insulin of the presentinvention further comprises the step of confirming that the silicananoparticles and branched polysaccharide are properly homogenized. Inanother embodiment, any of the following three tests are utilized: (a)the mixture appears homogenous; (b) the volume of the mixture is smallerthan the sum of volumes of the 2 components; and (c) the mixture doesnot sink when placed on the surface of a still body of water. In anotherembodiment, the composition reaches a minimum volume that is notdecreased upon further mixing. Each possibility represents a separateembodiment of the present invention.

The step of dry mixing is, in another embodiment, performed using a highshear mixer. In another embodiment, the step of mixing is performedusing a high-speed mixer. In another embodiment, the step of mixing isperformed using any other means suitable for generating a homogenoussolid phase from silica nanoparticles and a branched polysaccharide.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, step (a) of a method of manufacturing apharmaceutical composition for oral delivery of insulin of the presentinvention, i.e. the dry mixing step, further comprises inclusion of anadditional biopolymer that is a linear biopolymer. In anotherembodiment, the additional biopolymer is a linear polysaccharide. Inanother embodiment, the additional biopolymer is a linear high molecularweight structural protein. In another embodiment, the additionalbiopolymer is selected from the group consisting of chitin, cellulose, alinear alpha glucan, and a linear beta glucan. In another embodiment,the additional biopolymer is selected from the group consisting ofchitin, amylose, cellulose, and beta glucan. In another embodiment, aformulation method of the present invention comprises the steps ofincluding in the solid phase mixture both a branched polysaccharide anda linear polysaccharide. Formulation methods are provided herein thatcomprise inclusion of amylopectin, a branched polysaccharide, andchitin, a linear polysaccharide (Example 5). Other branchedpolysaccharides and linear polysaccharides disclosed herein are suitableas well. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the additional biopolymer of formulation methodsof the present invention is a dietary fiber, also known as “roughage.”In another embodiment, the dietary fiber is an insoluble fiber. Inanother embodiment, the dietary fiber is a linear insoluble fiber. Inanother embodiment, the dietary fiber is a soluble fiber. In anotherembodiment, the dietary fiber is a linear soluble fiber.

In another embodiment, a method of manufacturing a pharmaceuticalcomposition for oral delivery of insulin of the present inventioncomprises the steps of including a branched biopolymer, a linearpolysaccharide, and an insoluble fiber. In another embodiment, themethod comprises the steps of including in the particulate mattermixture a branched polysaccharide, a linear polysaccharide, and aninsoluble fiber. An example of such is a composition comprisingamylopectin, a branched polysaccharide; chitin, a linear polysaccharide;and cellulose, an insoluble fiber. Other branched and linearpolysaccharides and insoluble fibers disclosed herein are suitable aswell. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, a method of manufacturing a pharmaceuticalcomposition for oral delivery of insulin of the present inventionfurther comprises the step of adding an additional oil following theaddition of the first-added oil or mixture of oils. The term “additionaloil” encompasses an oil or mixture of oils, as described elsewhereherein. In another embodiment, the additional oil component comprises anantioxidant. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the insulin protein is included in the additionaloil or mixture of oils, instead of in the first-added oil or mixture ofoils.

In another embodiment, the additional oil, oil or mixture of oils has ahigher viscosity than the first-added oil or mixture of oils. In anotherembodiment, the use of a higher viscosity oil or oil mixture at thisstage enables formation of ordered structures in the composition.

In another embodiment, a method of manufacturing a pharmaceuticalcomposition for oral delivery of insulin of the present inventionfurther comprises the step of adding a third oil or mixture of oilsafter addition of the above-described additional oil or mixture of oils.In another embodiment, the third oil component comprises an antioxidant.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, a highly penetrative oil carrier is included inthe outer oil or mixture of oils. In another embodiment, the highlypenetrative oil carrier promotes efficient transport of the substancesinto the blood. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, a method of manufacturing a pharmaceuticalcomposition for oral delivery of insulin of the present inventionfurther comprises the step of adding a pharmaceutically acceptable waxfollowing the addition of the first-added oil or mixture of oils. Inanother embodiment, the wax is a substance with properties similar tobeeswax. In another embodiment, the wax is a substance having thefollowing properties: (a) plastic (malleable) at normal ambienttemperature; (b) having a melting point above approximately 45° C. (113°F.); (c) a low viscosity when melted, relative to a typical plastics;(d) insoluble in water; and (e) hydrophobic. In certain preferredembodiments, the wax is beeswax. In another embodiment, the waxstabilizes the matrix carrier composition. In another embodiment, theinclusion of wax facilitates formation of a tablet containing the matrixcarrier composition. Each possibility represents a separate embodimentof the present invention.

In another embodiment, the wax is heated as part of a method of thepresent invention. In another embodiment, the wax is pulverized. Inanother embodiment, the wax is both heated and pulverized. In anotherembodiment, the heating and/or pulverization are performed prior toblending with the other components. In another embodiment, the waxremains hot while blending with the other components is begun. Inanother embodiment, the heating and/or pulverization are performedduring blending with the other components. In another embodiment, theheating and/or pulverization are performed both prior to and duringblending with the other components. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, a composition of the present invention furthercomprises one or more pharmaceutically acceptable excipients, into whichthe matrix carrier composition is mixed. In another embodiment, theexcipients include one or more additional polysaccharides. In anotherembodiment, the excipients include one or more waxes. In anotherembodiment, the excipients provide a desired taste to the composition.In another embodiment, the excipients influence the drug consistency,and the final dosage form such as a gel capsule or a hard gelatincapsule.

Non limiting examples of excipients include: Antifoaming agents(dimethicone, simethicone); Antimicrobial preservatives (benzalkoniumchloride, benzelthonium chloride, butylparaben, cetylpyridiniumchloride, chlorobutanol, chlorocresol, cresol, ethylparaben,methylparaben, methylparaben sodium, phenol, phenylethyl alcohol,phenylmercuric acetate, phenylmercuric nitrate, potassium benzoate,potassium sorbate, propylparaben, propylparaben sodium, sodium benzoate,sodium dehydroacetate, sodium propionate, sorbic acid, thimerosal,thymol); Chelating agents (edetate disodium, ethylenediaminetetraaceticacid and salts, edetic acid); Coating agents (sodiumcarboxymethyl-cellulose, cellulose acetate, cellulose acetate phthalate,ethylcellulose, gelatin, pharmaceutical glaze, hydroxypropyl cellulose,hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate,methacrylic acid copolymer, methylcellulose, polyethylene glycol,polyvinyl acetate phthalate, shellac, sucrose, titanium dioxide,carnauba wax, microcrystalline wax, zein); Colorants (caramel, red,yellow, black or blends, ferric oxide); Complexing agents(ethylenediaminetetraacetic acid and salts (EDTA), edetic acid, gentisicacid ethanolmaide, oxyquinoline sulfate); Desiccants (calcium chloride,calcium sulfate, silicon dioxide); Emulsifying and/or solubilizingagents (acacia, cholesterol, diethanolamine (adjunct), glycerylmonostearate, lanolin alcohols, lecithin, mono- and di-glycerides,monoethanolamine (adjunct), oleic acid (adjunct), oleyl alcohol(stabilizer), poloxamer, polyoxyethylene 50 stearate, polyoxyl 35 casteroil, polyoxyl 40 hydrogenated castor oil, polyoxyl 10 oleyl ether,polyoxyl 20 cetostearyl ether, polyoxyl 40 stearate, polysorbate 20,polysorbate 40, polysorbate 60, polysorbate 80, propylene glycoldiacetate, propylene glycol monostearate, sodium lauryl sulfate, sodiumstearate, sorbitan monolaurate, sorbitan monooleate, sorbitanmonopalmitate, sorbitan monostearate, stearic acid, trolamine,emulsifying wax); Flavors and perfumes (anethole, benzaldehyde, ethylvanillin, menthol, methyl salicylate, monosodium glutamate, orangeflower oil, peppermint, peppermint oil, peppermint spirit, rose oil,stronger rose water, thymol, tolu balsam tincture, vanilla, vanillatincture, vanillin); Humectants (glycerin, hexylene glycol, propyleneglycol, sorbitol); Polymers (e.g., cellulose acetate, alkyl celluloses,hydroxyalkylcelluloses, acrylic polymers and copolymers); Suspendingand/or viscosity-increasing agents (acacia, agar, alginic acid, aluminummonostearate, bentonite, purified bentonite, magma bentonite, carbomer934p, carboxymethylcellulose calcium, carboxymethylcellulose sodium,carboxymethycellulose sodium 12, carrageenan, microcrystalline andcarboxymethylcellulose sodium cellulose, dextrin, gelatin, guar gum,hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, magnesium aluminum silicate, methylcellulose, pectin,polyethylene oxide, polyvinyl alcohol, povidone, propylene glycolalginate, silicon dioxide, colloidal silicon dioxide, sodium alginate,tragacanth, xanthan gum); Sweetening agents (aspartame, dextrates,dextrose, excipient dextrose, fructose, mannitol, saccharin, calciumsaccharin, sodium saccharin, sorbitol, solution sorbitol, sucrose,compressible sugar, confectioner's sugar, syrup); This list is not meantto be exclusive, but instead merely representative of the classes ofexcipients and the particular excipients which may be used in oraldosage compositions of the present invention. Each possibilityrepresents a separate embodiment of the present invention.

As provided herein, methods have been developed to formulate insulin inorally administrable form. The components are, in some preferredembodiments, mixed in a particular order in order to produce oil-coatedmatrix carrier compositions that protect the active ingredient fromdigestive processes in the stomach. Without wishing to be bound by anytheory or mechanism of action, the biopolymer, particularly whenbranched, absorbs hydraulic and mechanical stresses experienced duringdigestion. The oil coating constitutes a physical barrier that providesadditional protection against digestive enzymes. Secretion of bile acidstypically causes dispersion of the oil suspension into smallerparticles, which can be absorbed in the small intestine. While theparticle size is reduced after traversing the stomach and entering thesmall intestine, the particles remain in a size range of 30-1000 nm, toolarge to be a substrate for lipases and peptidases, preserving theprotective effect of the composition. Advantageously, lipid-coatingparticles of this size are absorbed to chylomicrons by lacteal vessels,which are lymphatic vessels originating in the villi of the smallintestine. Particles absorbed in this manner can reach the bloodstreamwithout undergoing first-pass metabolism, largely preserving thebiological activity of the insulin.

Matrix carriers for any insulin protein can be designed using thefollowing principles: For purposes of illustration, the followingformulas may be utilized in practicing the invention:

-   -   1. Quantify the R972 hydrophobic silica (specific area is about        110 m²*g⁻¹)

${Si} \geq {10*\frac{D_{IU}}{27.5}}$

-   -    wherein: D_(IU) is desired dosage of insulin per 1 ml in IU;        and Si is the concentration of silica mg*ml⁻¹        -   Note: 27.5 IU*mg⁻¹ is the specific activity of regular            insulin; the formula should be adjusted accordingly for            other types of insulin.    -   2. The specific weight of silica is about 2.4 g*cm⁻³, the        concentration of the branched polysaccharide in the medication        must therefore be at least 2.5 times of silica:

G _(AP)≧2.4*Si

-   -    Wherein: G_(AP) is the concentration of amylopectin.    -   3. The concentration of the linear polysaccharides (G_(LP)) is        estimated as:

$0.3 \leq \frac{G_{LP}}{G_{AP}} \leq 0.7$

Note:

Using the above mentioned concentration ratios between the silicananoparticles and the polysaccharides, ensures the stability of thepharmaceutical composition.

-   -   4. The chitin/fiber ratio may be between 0-1. High ratios (>0.5)        are used for the formation of fast term release insulin        compositions.    -   5. Thickness of the protective oil layer is at least 10 times        the diameter of a globular molecule or the maximal branch size        of an elongated molecule. The thickness of the oil coating of        the pharmaceutical compositions of the present invention is        determined by the following properties of the oil or mixture of        oils: (a) the viscosity and melting temperature; (b) the        acidity; and (c) the concentration of polar groups. Typically, a        first type of oil is chosen, preferably having a relatively low        viscosity and low concentration of polar groups. Suitable        examples are evening primrose oil, sesame oil, and silicon oil.    -   6. The concentration of insulin in the final formulation is        determined, based on its pharmacokinetics and pharmacodynamics.

The principles of the present invention are demonstrated by means of thefollowing non-limitative examples.

EXAMPLES Example 1 Preparation of Insulin Composition

An insulin composition (Formulation I) was produced, using the followingingredients:

Insulin Actrapid™, 9 ml

Olive oil, 11 ml

Benefiber™, 7 g

Silica R972, 1.2 g

Oblepicha, 9 ml

Sesame Oil up to 75 ml

Insulin was combined with sea buckthorn (oblepicha) oil and stirred at20 rpm for 2 min, and then at 50 rpm for 5 min. Benefiber™ (NovartisNutrition GmbH, Germany) and hydrophobic silica R972 were placed into abeaker and mixed by vortexing at 900 rpm for 5 min. Association betweenthe Benefiber™ and the silica was determined by the mixture's ability tofloat after being placed on the surface of a water-filled beaker. TheBenefiber™/silica mixture was added to the oil-insulin solution andstirred for 25 minutes at 50 rpm. Olive oil was added, and the mixturewas stirred at 50 rpm for 3 min. The volume was brought up to 75 ml withsesame oil, and the mixture was stirred at 50 rpm for 20 min. Theproduct was stored refrigerated (3-8° C.). The final insulinconcentration was 12 IU/ml. For animal experiments, the composition wasadministered by gavage. Other preparation of the product were packagedinto gelatin enteric covering capsules.

Example 2 Additional Actrapid™ Matrix Carrier Composition Formulated forShort-Life

An additional Actrapid™ formulation (Formulation II) using the followingingredients was designed for short-term insulin release:

Insulin Actrapid™, 1 ml

Olive oil, 1.5 ml.

Ambrotose™, 0.7 g.

Silica R972, 0.1 g

Oblepicha oil, 1.5 ml

Evening primrose oil, 5 ml

0.7 g of rice polysaccharides (Ambrotose™, Mannatech Inc, Coppell, Tex.75019, USA) was combined with 0.1 g hydrophobic fumed silica R972(Degussa Inc), and mixed by vortexing at 900 rpm for 5 min. Associationbetween the Ambrotose™ and the silica was determined by the mixture'sability to float after being placed on the surface of a water-filledbeaker 1 ml Actrapid™ insulin were added and stirred for 15 minutes at50 rpm. 1.5 ml of olive oil was added and stirred for 2 minutes at 100rpm with a magnetic stirrer. Sea buckthorn (oblepicha) oil was added andstirred for 2 minutes at 100 rpm with a magnetic stirrer. The volume wasbrought up to 5 ml with evening primrose oil and stirred at 50 rpm for20 min. The product was stored refrigerated (3-8° C.). In a separatepreparation, the amount of ingredients used was doubled, yieldingidentical results.

The final insulin concentration was 20 IU/ml. For human administration,the product was packaged into 25 gelatin enteric covering capsules.

In additional experiments, vitamin E was included in any one of the oilsused.

Example 3 Longterm Release Actrapid™ Matrix Carrier Composition

The following formulation (Formulation III) was manufactured to providelonger-term Actrapid™ release:

Olive oil, 10 ml.

Benefiber™, 1.5 g.

Insulin Actrapid™, 2 ml

Silica R972, 0.7 g

Oblepicha oil, 10 ml

Evening primrose oil, 5 ml

Linseed oil, up to 40 ml.

Benefiber™ was combined with hydrophobic fumed silica R972 and mixed byvortexing at 900 rpm for 5 minutes. Association between the Benefiber™and the silica was determined by the mixture's ability to float afterbeing placed on the surface of a water-filled beaker. Actrapid™ insulinwas added and stirred for 15 minutes at 50 rpm. Evening primrose oil wasadded and stirred for 2 minutes at 100 rpm with a magnetic stirrer. Seabuckthorn (oblepicha) oil was added and stirred for 2 minutes at 100 rpmwith a magnetic stirrer. Olive oil was added and stirred for 2 minutesat 100 rpm with a magnetic stirrer. The volume was brought up to 40 mlwith olive oil and stirred at 50 rpm for another 20 min. The product wasstored refrigerated (3-8° C.).

The final insulin concentration was 5 IU/ml. For human administration,the product was packaged into 25 gelatin enteric covering capsules(commercially available from Shionogi and Company, Ltd, Japan)containing 1.6 ml=7.8 IU insulin each.

Example 4 Preparation of Additional Actrapid™ Matrix Carrier Composition

An additional Actrapid™ matrix carrier composition (Formulation V) wasproduced, using the following ingredients:

Olive oil, 11 ml

Benefiber™, 3 g

Insulin Actrapid™, 9 ml

Oblepicha oil, 9 ml

Hydrophobic silica R972, 1.2 g

Sesame Oil up to 75 ml.

Benefiber™ (Novartis Nutrition GmbH, Germany) and silica were placedinto a beaker and mixed by vortexing at 900 rpm for 5 minutes.Association between the Benefiber™ and the silica was determined by themixture's ability to float after being placed on the surface of awater-filled beaker. Actrapid™ insulin was added and stirred for 15minutes at 50 rpm. Sesame oil and sea buckthorn (oblepicha) oil werecombined in a beaker and vortexed on a low setting for 15 minutes. Oliveoil was added to the oils and stirred with a glass rod. The solid phasemixture and oil mixture were combined and mixed at 100 rpm with amagnetic stirrer. The volume was brought up to 75 ml with sesame oil andstirred with a glass rod. The product contained 12 IU/ml insulin and waspackaged into gelatin enteric covering capsules.

An additional insulin matrix carrier composition (Formulation VI) wasprepared using NovoRapid™ insulin, using the above protocol. In thiscase NovoRapid™ insulin was used instead of Actrapid™ insulin.

Example 5 Additional Insulin Matrix Carrier Composition

An additional insulin matrix carrier composition (Formulation IV) wasprepared using BIOCON insulin, using the following protocol and theingredients set forth in Table 1, using methods similar to those setforth in previous Examples. A light microscopy picture of thecomposition is shown in FIG. 3.

1. Mix oblepicha+olive oil+1/3 of sesame oil.2. Add Insugen™ insulin powder (BIOCON) into the mixture of oils andmix.3. Mix fiber+chitin+amylopectin+silica.4. Add the mixture of step 3 to the mixture of oils and insulin of step2 and mix.5. Add the rest of the sesame oil and mix.

TABLE 1 Ingredients for the preparation of the BIOCON matrix carriercomposition. Formulation IV Insulin Powder (BIOCON), mg 36.4 109.2 182Olive Oil, ml 33 33 33 Sea Buckthorn Oil (Oblepicha), ml 42 42 42 SesameOil, ml up to 100 up to 100 up to 100 Amylopectin, g 11.25 11.25 11.25Chitin, g 1.9 1.9 1.9 Silica R972, g 2.5 2.5 2.5 Final concentration ofinsulin, IU/ml 10 30 50

Next, an additional short-term release insulin matrix carriercomposition (Formulation A) was prepared using BIOCON insulin, using theingredients set forth in Table 2.

TABLE 2 Ingredients for the preparation of an insulin matrix carriercomposition (Formulation A). Ingredient Amount Olive oil 20 mlAmbrotose* 3 g Insulin powder 70 mg Silica R972 0.6 g Sea buckthorn(oblepicha) oil 26 ml Sesame oil up to 70 ml Ambrotose ™ powder containsArabinogalactan (a gum from the Larix decidua tree), Manapol, which is agel extracted from the inner leaf of aloe vera gel plant, gum ghatti,and gum tragacanth or Manapol powder, oat fiber, brown macroalgae(Undaria pinnatifida) sporophyll, vegetarian glucosamine-HCl, ghattigum, gum tragacanth and xylitol.

Example 6 Efficacy of the Oral Insulin Composition of the PresentInvention in Diabetic Mice Materials and Experimental Methods

Streptozotocin (STZ)-induced diabetes treatment: Diabetes was induced by2 injections of 500 and 700 μl of 1.5 mg/ml streptozotocin separated by48 hr, in male adult BALB/c mice (7-10 weeks old) of an average weightof 23-28 gr. Untreated mice of approximately the same age and weightwere used as control. Blood glucose levels (BGL) were assessed 48 hoursafter STZ injection by a standard FreeStyle™ glucometer (Abbot DiabetesCere Inc, Alameda, Calif.) from the tail vein blood samples.

Insulin compositions were administered orally to mice by gavage (1 mlvolume), without prior deprivation of food or water. During theexperiment mice were supplied with food and water as usual.

Compositions: The first experiment utilized Formulations V and VIdescribed in Example 4. The second experiment utilized Formulation IVdescribed in Example 5.Blood insulin concentration. Blood insulin concentrations were detectedby ELISA (Human Insulin ELISA kit, Linco).

Treatment Groups:

1. Control group 1: no STZ treatment, no insulin administration.2. Control group 2: no STZ treatment, oral insulin composition of thepresent invention administered by gavage3. Control group 3: STZ treated, no insulin administration.4. Diabetic (STZ treated) mice; insulin administered by SC injection.5. Diabetic (STZ treated) mice; insulin was administered by gavage.6. Diabetic (STZ treated) mice; insulin was administered using the oralinsulin composition of the present invention by gavage.7. Diabetic (STZ treated) mice; matrix carrier (without insulin)administered by gavage as control.

Results

In a first experiment, diabetes was induced by streptozotocin (STZ) inmale adult BALB/c mice, followed by administration of NovoRapid™ (9.5IU) and Actrapid™ (12 IU) based insulin compositions of the presentinvention. Both compositions significantly reduced blood glucose levels(FIGS. 4A-B, respectively). By contrast, STZ-treated mice that receivedempty matrix carrier compositions (lacking insulin), orally administeredActrapid™ or NovoRapid™ insulin, or were given 25 IU Insulin (BIOCON) inPBS (gavage) (FIG. 5) did not exhibit significant reduction in bloodglucose levels. Normal mice (not STZ-treated) that received insulincompositions exhibited no significant reduction in blood glucose level.Normal and diabetic mice injected with insulin, by contrast, exhibitedhypoglycemia symptoms that were in some cases fatal.

In a second experiment, an insulin composition of the present inventionwas administered orally to STZ-treated mice by gavage (1 ml volume) indosages ranging from 2-10 IU. A dose-responsive reduction in bloodglucose levels was observed for 9-12 hours; however, levels rarelydropped below 100 mg/dL (FIG. 6A-D). The presence of human insulin inthe blood following administration of the insulin matrix carriercomposition was confirmed by ELISA. By contrast, subcutaneous injectionof 10 IU of insulin caused near-fatal hypoglycemia (FIG. 6E). Normalmice receiving 2, 5, or 10 IU insulin compositions exhibited only aslight reduction in blood glucose level (FIG. 6F), while those receivinginjected insulin experienced a precipitous and occasionally fatal dropin glucose levels. As before, STZ-treated mice that received emptymatrix carrier compositions (lacking insulin), orally administeredinsulin, or were left untreated did not exhibit significant bloodglucose level reduction.

In another experiment, direct comparison of 10, 5, or 2 IU of insulinmatrix carrier composition (Formulation IV) vs. injection of the sameamount of insulin solution (a standard formulation) in 14-25 g micereveled that mice treated with the oral insulin composition of thepresent invention maintained normal blood glucose levels for longerperiods of time compared to the insulin injected mice. These observationreflect on the increased bioavailability of insulin when administeredwithin the matrix carrier composition of the present invention. Inaddition, mice administered the matrix carrier compositions had nohypoglycemia, while the injected mice exhibited severe hypoglycemia(FIGS. 6G, H, I for 10, 5, and 2 IU, respectively).

FIGS. 6J-K: We have compared the pharmacodynamics of Formulation A(Example 5) and. Formulation IV (Example 5). Results are described inFIG. 6J (2 IU of insulin) and FIG. 6J (7.5 IU of insulin) according towhich mice administered Formulation IV showed lower blood glucose levelsfor longer periods of time as opposed to mice administered FormulationA.

Indication for the increased bioavailability of the insulin administeredorally using the compositions of the present invention in comparisonwith injected insulin can be found by calculating the “Effective Areas”.“Effective Area” is defined as the sum of the net changes in bloodglucose level (BGL) values relative to the basal level, along a definedperiod of time, calculated as follows:

1. Obtain a baseline average of BGL for each time point.2. For each time point, subtract the BGL value in the treated groups(oral insulin of the present invention and injected insulin) from thebaseline average.3. Sum the values obtained in step 2 for all time points.To obtain the values in the table, the “effective areas” of thedifferent treatments were then subtracted or divided. Results aredepicted in Table 3.

TABLE 3 Effective Areas of insulin oral matrix carrier compositionsversus injected insulin. Group Value 10 IU: (injection- matrix carriercomposition) −1042.7 mg/dL   5 IU: (injection- matrix carriercomposition) 340.29 mg/dL 2 IU: (injection- matrix carrier composition) 834.4 mg/dL 10 IU: injection/matrix carrier composition 0.64 5 IU:injection/matrix carrier composition 1.32 2 IU: injection/matrix carriercomposition 3.73 10 IU injection/5 IU injection 1.32 10 IU injection/2IU injection 1.61 10 IU matrix carrier composition/5 IU matrix 2.74carrier composition 10 IU matrix carrier composition/2 IU matrix 9.45carrier composition

As shown in Table 2, the relatively low 10 IU/5 IU and 10 IU/2 IU ratiosfor injected insulin indicate that these doses are approaching thesaturation dose for the mice. By contrast, the relatively large ratiosfor the insulin matrix carrier composition indicate that it is far fromthe saturation doses. Thus, matrix carrier compositions of the presentinvention are more amenable to accurate dosing within their therapeuticrange, compared with standard injected insulin formulations.

Example 7 The Efficacy of the Oral Insulin Compositions of the PresentInvention in Human Subjects

The effect of an insulin composition of the present invention was testedon two human subjects, one healthy and one diabetic. 30 IU of Actrapid™matrix carrier composition (Formulation II) reduced blood glucose levelsin the healthy subject from 105 to 80-90 mg/dL over a six-hour testperiod (FIG. 7A). The subject reported an unusual degree of hunger, butotherwise no adverse reactions. By contrast, administration of injectedinsulin to healthy subjects is known to cause hypoglycemia, in somecases severe, with accompanying adverse reactions.

10 IU of the same formulation V (Example 4) were administered 3 timesper day over 14 days to a 67-year-old subject having type I/II diabetes,who exhibited glucose levels of over 170 when untreated and 130-170 whenreceiving Gluco-Rite™. Upon taking the oral insulin composition of thepresent invention the blood glucose levels dropped to an average ofabout 130 (FIG. 7B). The subject reported feeling well during the entireperiod of receiving the oral insulin composition of the presentinvention. The subject has continued to take the compositions 3-4 timesper day, as needed, resulting in well-controlled glucose levels with noadverse reactions. By contrast, the subject had a long-standing historyof intense sensations of dread and unease after receiving a number ofdifferent injected insulin formulations.

The presence of elevated insulin levels in the diabetic subject's bloodfollowing administration of the insulin composition was confirmed byELISA.

Thus, insulin compositions of the present invention are capable oforally delivering various forms of insulin in a biologically active formthat can effectively treat diabetes. They have the additional advantageof not inducing hypoglycemia in either diabetic or normal subjects.

Example 8 Toxicity Study of Chronic Oral Administration of InsulinCompositions Materials and Experimental Methods

Fifteen 10 week-old Balb/C male mice were used. Mice were administereddaily 1 ml of insulin matrix carrier composition (25 IU/ml)(experimental group) or PBS (gavage control group) via gavage over 15days. On the 14th and 15th day, mice were administered orally 100 ng oflipopolysaccharides (LPS) together with the insulin matrix carriercomposition or PBS. Negative control mice were administered 1 ml PBS bygavage over 13 days, and 100 ng of LPS in 1 ml PBS by gavage on the 14thand 15th day. Positive control mice were injected with 1 mg of LPS in 1ml of PBS. 3 h after LPS administration, mice were sacrificed, blood wascollected (for LPS detection) and gastro-system, liver and kidneys werefixed in paraformaldehyde (PFA) 4% for histological analysis.

Animal follow up and macroscopic analysis: Mice were weighed every 3days, and their fur condition was detected daily. After sacrifice, allorgans or tissues were investigated for the presence of pathologicalchanges.Internal organs collection and fixation: On day 15 mice were sacrificed,and their gastro-system, kidney, and liver were collected from theabdominal cavity, weighed and fixed in 10% formalin solution.Blood collection and plasma preparation: Blood was collected from theheart of the mice into tubes that contain EDTA. Plasma was be separatedfrom the blood by centrifugation; at first for 15 min at 3000×gmaxfollowed by 15 min at 16,000×gmax. Supernatant was removed, placed in anew tube and stored at −20° C.Detection of LPS in mouse serum: LPS in mouse serum was detected byHPLC. Sera taken on day 15 from the groups described above were preparedfor HPLC analysis by addition of 0.1M of EDTA.

Results

The toxicity of chronic administration of oral insulin compositions ofthe present invention was investigated. No pathological changes wereobserved in the animals' behavior. Their fur was in normal condition,appearing smooth, clean, and bright. No weight loss was detected; themice gained weight normally.

Microscopic and Macroscopic Organ Analysis:

Microscopic analysis showed no evidence of pathology in tissues of micein all groups (liver—FIG. 8A; kidney—FIG. 8B; duodenum—FIG. 8C). Inaddition, macroscopic analysis of internal organs revealed no evidenceof any pathology. Organs of all mice were normally developed, had normalsize, shape, appearance (bright and smooth), and weight, were normallycolored, and were in their normal location. Livers were in all casessituated under diaphragm and smooth and bright, and weights were about1.6 g. Liver parenchyma were dark colored. Kidneys exhibited a smoothsurface. Weight was consistently about 0.2 gr. Esophagus, stomach, andlarge and small intestine exhibited normal size and location, andpillories and duodenum were open.

Serum from the treated and untreated mice were also tested for thepresence of LPS. LPS was not present in serum of mice given oral insulincomposition of the present invention+LPS nor in serum of mice given 1 mlPBS+LPS, showing that neither the matrix carrier composition nor thegavage compromised the integrity of the mice's gastrointestinal linings.Serum from mice injected 1 mg of LPS in 1 ml of PBS served as a positivecontrol, and untreated mice served as negative control.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed functions may take avariety of alternative forms without departing from the invention.

1-45. (canceled)
 46. A pharmaceutical composition for oral usecomprising an oil having particulate matter suspended therein, whereinthe particulate matter comprises: a. a polysaccharide in intimatenon-covalent association with silica nanoparticles having a hydrophobicsurface, wherein the size of the silica nanoparticles is between 1-100nanometers; and b. an insulin protein non-covalently associated withsaid silica nanoparticles and the polysaccharide.
 47. The pharmaceuticalcomposition of claim 46, wherein the polysaccharide comprises a branchedpolysaccharide.
 48. The pharmaceutical composition of claim 46, whereinsaid polysaccharide comprises amylopectin, starch, glycogen, chitin,cellulose, amylose, beta glucan, and combinations and derivativesthereof.
 49. The pharmaceutical composition of claim 46, wherein saidcomposition is anhydrous.
 50. The pharmaceutical composition of claim46, wherein said size of said silica nanoparticles is between 5-30nanometers.
 51. The pharmaceutical composition of claim 46, wherein saidhydrophobic surface of said silica nanoparticles comprises hydrocarbonmoieties.
 52. The pharmaceutical composition of claim 46, furthercomprising an additional biopolymer selected from the group consistingof a polysaccharide and a high molecular weight structural protein,wherein said additional biopolymer is a linear biopolymer.
 53. Thepharmaceutical composition of claim 52, wherein said additionalbiopolymer is a high molecular weight structural protein selected fromthe group consisting of elastin, collagen, keratin, fibrinogen andcombinations and derivatives thereof.
 54. The pharmaceutical compositionof claim 46, wherein said oil comprises a mixture of oils selected fromnatural vegetable oils and synthetic analogues thereof.
 55. Thepharmaceutical composition of claim 46, wherein said composition furthercomprises an antioxidant.
 56. The pharmaceutical composition of claim46, further comprising a wax.
 57. The pharmaceutical composition ofclaim 46 for administering insulin to a subject.
 58. The pharmaceuticalcomposition of claim 46 for treating diabetes in a subject.
 59. A methodof manufacturing a pharmaceutical composition formulated for oraldelivery of insulin, said method comprising the steps of: a. mixingsilica nanoparticles having a hydrophobic surface, wherein the size ofsaid silica nanoparticles is between 1-100 nanometers, with apolysaccharide, whereby said silica nanoparticles form an intimatenon-covalent association with said polysaccharide; b. mixing an insulinprotein with an oil; and c. mixing said silica nanoparticles andpolysaccharide into the oil, wherein said insulin forms an intimatenon-covalent association with said silica nanoparticles and saidpolysaccharide and wherein said silica nanoparticles, saidpolysaccharide and said insulin protein are dispersed in said oil. 60.The method of claim 59, wherein the polysaccharide comprises a branchedpolysaccharide.
 61. The method of claim 59, further comprising the stepof adding an additional oil component following the addition of the oil.62. The method of claim 59, further comprising the step of adding a waxfollowing the addition of said oil.
 63. The method of claim 59, furthercomprising the step of adding an additional biopolymer to the mixture ofsilica nanoparticles and polysaccharide, wherein said additionalbiopolymer is a linear biopolymer.
 64. A method of manufacturing apharmaceutical composition formulated for oral delivery of insulin, saidmethod comprising the steps of: a. mixing silica nanoparticles having ahydrophobic surface, wherein the size of said silica nanoparticles isbetween 1-100 nanometers, with (a) a polysaccharide, and (b) an insulinprotein, whereby said silica nanoparticles form an intimate non-covalentassociation with said polysaccharide and said insulin protein; and b.mixing said silica nanoparticles, said polysaccharide, and said insulinprotein into an oil, wherein said silica nanoparticles, saidpolysaccharide, and said insulin protein are suspended in said oil. 65.The method of claim 64, wherein the polysaccharide comprises a branchedpolysaccharide.
 66. The method of claim 64, further comprising the stepof adding an additional oil component following the addition of the oil.67. The method of claim 64, further comprising the step of adding a waxfollowing the addition of said oil.
 68. The method of claim 64, furthercomprising the step of adding an additional biopolymer selected from thegroup consisting of a polysaccharide and a high molecular weightstructural protein, wherein said additional biopolymer is a linearbiopolymer.
 69. The method of claim 64, wherein said insulin is in a drylyophilized form.